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Cite this: DOI: 10.1039/c5cc02069g Received 12th March 2015, Accepted 23rd March 2015
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A plasmonic aptasensor for ultrasensitive detection of thrombin via arrested rolling circle amplification† Sai Wang, Sai Bi,* Zonghua Wang,* Jianfei Xia, Feifei Zhang, Min Yang, Rijun Gui, Yanhui Li and Yanzhi Xia
DOI: 10.1039/c5cc02069g www.rsc.org/chemcomm
A sensitive signal generation mechanism for gold nanoparticle growth by reducing gold ions with hydrogen peroxide is applied in a plasmonic aptasensor, achieving naked-eye detection of thrombin at the singlemolecule level based on the specific interaction of aptamer–thrombin via an arrested rolling circle amplification to yield horseradish peroxidase (HRP)-mimicking DNAzymes as biocatalysts.
Colorimetric methods have become attractive tools for biomolecule detection due to their simplicity, cost-effectiveness, and not needing sophisticated equipment.1 However, conventional colorimetric detection methods, such as the enzyme-linked immunosorbent assay (ELISA), are often based on the differentiation of two similar looking lightly colored solutions using organic compounds as substrates (e.g., 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB), 2,2 0 azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), o-phenylenediamine (OPD)), which cannot facilitate the confident detection of target molecules at low concentrations.2 Alternatively, plasmonic nanoparticle-based visual detection can potentially improve the standard of colorimetric methods,3 which are often based on target-induced aggregation of functionalized gold nanoparticles (AuNPs) with the easy observation of different colored solutions of either blue or red.4 In these assays, the spherical AuNPs had to be synthesized previously and then required specific storage and reaction conditions, especially salt concentration, to generate a colored signal.5 In addition, it is still difficult to meet the sensitivity requirements for the detection of analytes at ultralow concentrations. To address these issues, Rica and Stevens reported a plasmonic nanosensor for antigen detection down to 40 zM by means of an enzyme-guided crystal growth of gold nanostars.6 Very recently, the College of Chemical Science and Engineering, 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, P. R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: Experimental section and additional results. See DOI: 10.1039/c5cc02069g
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same group also developed a plasmonic ELISA for the detection of antigens at ultralow concentrations (1018 g mL1) with the naked eye.7 Although these plasmonic methods achieved ultrahigh sensitivity, protein enzymes, such as glucose oxidase (GOx) or catalase, were used to control the concentration of hydrogen peroxide, but these methods are often hampered by their intrinsic drawbacks, such as low operational stability, high cost of preparation and purification, sensitivity of catalytic activity to environmental conditions and difficulty of modification.8 As a promising candidate, a HRP-mimicking DNAzyme with high catalytic properties has the advantages of easy synthesis and modification with low cost, as well as an impressive stability against stringent conditions. To date, DNAzymes have been widely applied as biolabels that transduce and amplify sensing events.9 More importantly, to achieve a higher detection sensitivity, innovative amplification strategies can be incorporated into these plasmonic methods. Rolling circle amplification (RCA) is an isothermal enzymatic process to generate long single-stranded DNA or RNA using a short strand of DNA or RNA as a primer and circular DNA as a template in the presence of unique polymerases.10 Due to its simplicity and versatility, RCA has been explored extensively for the sensitive detection of various targets.11 Importantly, functional sequences (e.g., HRP-mimicking DNAzymes) can be readily incorporated into RCA products by designing the template, and can act as self reporters to generate an optical or electrochemical signal.12 In addition, aptamers are single-stranded oligonucleotides that are selected by the systematic evolution of ligands by exponential enrichment (SELEX) and have emerged as alternative biorecognition elements with a high specificity and affinity for a variety of targets ranging from metal ions and small molecules to proteins, viruses, and even cells.13 Recently, Fan’s group has found that the aptamer–thrombin complex could prevent Klenow DNA polymerase from reading through the aptameric domain, which would indirectly inhibit in vitro replication of DNA.14 Li et al. further exploited the aptamer–protein interactions that can arrest the RCA reaction using phi29 DNA polymerase with a circular DNA template.15
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Fig. 1 Native PAGE of RCA products in the presence (lane 1) and absence (lane 2) of thrombin. The concentrations of the primer, circular template and thrombin are 5 mM. M represents the marker.
Scheme 1 (a) Schematic illustration of the visual and ultrasensitive detection of thrombin using a plasmonic aptasensor based on arrested RCA. (b) Growth mechanism of AuNPs. Arrows represent the 30 -terminus of DNA strands.
Herein, we propose a plasmonic aptasensor for the naked eye detection of thrombin with ultrahigh sensitivity on the basis of arrested RCA. The principle of the strategy is presented in Scheme 1. To demonstrate the proof of principle, we chose thrombin, the central protease in the blood coagulation cascade proteases, as the target analyte of interest.16 Firstly, amino-modified DNA (1) is covalently immobilized on each well of a microtiter plate using glutaraldehyde as the coupling reagent. The coverage of DNA (1) on each well was calculated to be 9.68 pmol per well (see ESI†). A padlock DNA probe (2), consisting of five regions, is used as the circular template for the subsequent arrested RCA. Regions I and II (black, 10 mer) are complementary to the half sequence of (1); regions III and IV (blue, 17 mer) are complementary sequences to the HRP-mimicking DNAzymes; region V (purple, 15 mer) is the aptamer segment, which can bind to the fibrinogen-binding site of a-thrombin through the formation of a G-quadruplex structure with a high affinity and specificity (dissociation constant (Kd) = 25 nM).15 Therefore, the 50 - and 30 -end of the padlock probe (2) can be ligated and circularized through hybridizing regions I and II with DNA (1) in the presence of T4 DNA ligase to obtain the aptamer circular template. Subsequently, the addition of thrombin stimulates the self-assembly of region V in (2) into a tight aptamer– thrombin complex, making the circularized (2) unsuitable as a template for phi29 DNA polymerase and resulting in the prohibition of RCA. We refer to this process as arrested RCA. In contrast, in the absence of thrombin, the RCA reaction can be initiated in the presence of phi29 DNA polymerase/dNTPs by using the singlestranded DNA (1) as a primer and the circular padlock probe (2) as a template. The resultant RCA products containing multiple complementary sequences to the circle template are generated, further forming a large number of hemin/G-quadruplex HRP-mimicking DNAzymes in the presence of hemin. The reaction pathways of RCA with the aptamer circular template were verified by native polyacrylamide gel electrophoresis (PAGE) (Fig. 1). In the absence of thrombin, the aptamer circular template serves as the regular circular template for the RCA reaction in the presence of phi29 DNA polymerase/dNTPs, resulting in the generation of long strand DNA products that are too large to enter the gel by electrophoresis (lane 2). Conversely, no RCA product can be observed from the sample in the presence of
Chem. Commun.
thrombin, reliably confirming that thrombin specifically engages the aptamer into a fairly stable complex that arrests RCA by inhibiting the strand-displacement ability of phi29 DNA polymerase (lane 1). As shown in Scheme 1b, in the proposed plasmonic aptasensor we apply the hemin/G-quadruplex HRP-mimicking DNAzymes as biocatalytic labels that are linked to the growth of AuNPs to obtain blue- or red-colored solutions in the presence or absence of thrombin. In the presence of thrombin, hydrogen peroxide can reduce the gold precursor at a fast rate in the 2-(N-morpholino)ethanesulfonic acid (MES) buffer, resulting in the formation of quasi-spherical and well-dispersed AuNPs with a red color. In the absence of thrombin, the HRP-mimicking DNAzyme catalyzes the decomposition of hydrogen peroxide into H2O and O2, which slows down the kinetics of crystal growth. As a result, aggregated AuNPs with ill-defined morphology are formed, showing a blue color. Since it is easy to distinguish the two differently colored solutions (blue and red) at a glance, the plasmonic method allows ultrasensitive detection of thrombin with the naked eye. In this reaction, for the growth of AuNPs, the concentration of hydrogen peroxide is a critical condition that can affect the morphology and optical properties of AuNPs, therefore different concentrations of hydrogen peroxide were added to a 0.1 mM Au3+ solution in the presence of MES to act as a supporting reducing agent and a capping ligand.17 As shown in Fig. 2a, the tonality of the solution changes from blue to red gradually as the concentration of hydrogen peroxide increases in the range from 1 to 11 mM after reacting for 15 min. The results indicate that when the concentration of hydrogen peroxide is low, clusters of aggregated AuNPs are formed, which generates a blue color. Nevertheless, when hydrogen peroxide is at a high concentration, non-aggregated
Fig. 2 (a) Photograph and (b) UV-vis spectra showing AuNP growth after adding different concentrations of hydrogen peroxide to a growth solution containing 0.1 mM Au3+ in 1 mM MES buffer (pH 6.5). (c) UV-vis absorbance at 540 nm corresponding to different concentrations of hydrogen peroxide.
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colloidal suspensions are obtained, and the solution can be easily identified by a red color. The color change corresponds to a wavelength shift in the surface plasmon resonance of AuNPs, which was verified by UV-vis spectra (Fig. 2b). As the concentration of hydrogen peroxide decreases, the wavelength of the peak absorbance is red-shifted (see ESI,† S3), indicating the formation of aggregated AuNPs at low concentrations of hydrogen peroxide. Furthermore, AuNP growth for different concentrations of hydrogen peroxide was investigated by measuring the UV-vis absorbance at 540 nm (Fig. 2c), showing a critical point when the concentration of hydrogen peroxide is 3 mM. Overall, in this study the morphology and state of aggregation of AuNP growth is sensitive to the concentration of hydrogen peroxide as it acts as reducing agent to reduce gold ions in MES according to eqn (1) in Scheme 1b. Moreover, according to eqn (2) in Scheme 1b, the HRPmimicking DNAzyme can catalyze the decomposition of hydrogen peroxide into H2O and O2. The influences of the hemin/ G-quadruplex HRP-mimicking DNAzyme and hemin on AuNP growth were also investigated and the high catalytic activity of HRP-mimicking DNAzymes to reduce the concentration of hydrogen peroxide that directly influences the growth of AuNPs has been demonstrated (see ESI†). Subsequently, we readily adapted the sensitive detection system to biosensing strategies by using HRP-mimicking DNAzymes as labels to control the concentration of hydrogen peroxide, and further improved the naked-eye detection of the analytes at low concentrations by combining with the amplification protocol of arrested RCA as illustrated in Scheme 1. Fig. 3a shows the results for the naked-eye detection of thrombin with the proposed plasmonic aptasensor. As the concentration of thrombin increases, the red color intensifies. This observation is attributed to the decreased generation of HRP-mimicking DNAzymes via the RCA reaction as a result of the specific binding between thrombin and the aptamer in
Fig. 3 Photograph (a) and corresponding UV-vis calibration curve (b) for thrombin detection with the plasmonic aptasensor. (c and d) TEM images of AuNPs grown by the plasmonic aptasensor in the absence and presence of thrombin (1 fM), respectively. The final concentrations of hydrogen peroxide and Au3+ in the MES buffer (1 mM, pH 6.5) are 3 mM and 0.1 mM, respectively.
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circular DNA that is thus unsuitable as the template for the phi29 DNA polymerase; this increases the concentration of hydrogen peroxide and favors the generation of red solutions of AuNPs. Remarkably, the color obtained by 1017 M thrombin could be easily differentiated from the blank (thrombin concentration of 0), correspondingly achieving a limit of detection as low as B6 molecules in a 1 mL test sample. Moreover, from the calibration curve in Fig. 3b, the UV-vis absorbance at 540 nm increases with the increase in thrombin concentration, which is consistent with the color of te nanoparticle solutions observed in Fig. 3a. The ultrahigh sensitivity is attributed not only to the detection system of AuNP growth that is extremely sensitive to the concentration of hydrogen peroxide, but also to the efficient amplification of RCA and the high catalytic action of the HRPmimicking DNAzyme that has multiple turnovers through RCA. The morphology of the resultant colloidal solutions in the absence and presence of thrombin were further confirmed by TEM, and were grown as clusters of aggregated and nonaggregated AuNPs, respectively, as expected (Fig. 3c and d). Therefore, these results demonstrate the feasibility of the plasmonic aptasensor for the ultrasensitive detection of the target thrombin that controls the generation of HRP-mimicking DNAzymes via arrested RCA and further regulates the growth of AuNPs with a desired state of aggregation to yield a colored solution with a distinct tonality. Moreover, we challenged the plasmonic aptasensor with four non-cognate proteins: bovine serum albumin (BSA), bovine hemoglobin (BHb), lysozyme and nucleolin. From Fig. 4, the red color is only observed for thrombin, whereas the other samples yield blue-colored nanoparticle solutions even though the analytes are at a concentration of 1 fM. This excellent specificity is attributed to the direct concentration relationship between the HRP-mimicking DNAzyme and thrombin, based on the high selectivity and affinity of the aptamer in the circular template to thrombin for the performance of the RCA reaction. In conclusion, we have adapted the sensitive naked-eye detection system of AuNP growth through the reduction of gold ions with hydrogen peroxide as a signal generation mechanism for the fabrication of a plasmonic aptasensor. To further improve the detection sensitivity, the amplification technique of RCA is incorporated to generate a cascade of HRP-mimicking DNAzymes as biocatalysts to control the concentration of hydrogen peroxide. By rationally encoding an aptamer sequence into the circular template, an arrested RCA strategy is proposed based on the inability of phi29 DNA polymerase to read through the aptamer–thrombin complexes. Significantly, this plasmonic aptasensor achieves an ultralow detection limit of 10 aM thrombin (B6 molecules in a 1 mL test sample),
Fig. 4 Photographs for the detection of various analytes with the plasmonic aptasensor. The concentration of each analyte is 1 fM. The reaction conditions are the same as those in Fig. 3.
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which greatly improves upon the sensitivity of traditional colorimetric methods using AuNPs (ESI,† S5) and other RCA strategies for thrombin detection (ESI,† S6), giving great promise as a versatile tool for single-molecule detection in biosensing and biomedicine. This work was supported by the National Science Foundation of China (21375056, 21405086, 21475071, 21275082), the Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-12-1024), the Taishan Scholar Program of Shandong Province, the Natural Science Foundation of Shandong (ZR2014BQ001, BS2014NJ023) and the Natural Science Foundation of Qingdao (13-1-4-128-jch and 13-1-4-202-jch).
Notes and references 1 C. Feng, S. Dai and L. Wang, Biosens. Bioelectron., 2014, 59, 64–74. 2 Z. Fang, B. Jiang, W. Wu, Z. Xiang, C. Ouyang, T. huang, J. Chen and L. Zeng, Chem. Commun., 2013, 49, 6164–6166. 3 X. Xie, W. Xu and X. Liu, Acc. Chem. Res., 2012, 45, 1511–1520. 4 W. Zhao, M. A. Brook and Y. Li, ChemBioChem, 2008, 9, 2362–2371. 5 H. Li and L. J. Rothberg, J. Am. Chem. Soc., 2004, 126, 10958–10961.
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ChemComm ´lvarez-Puebla, L. M. Liz6 L. Rodrı´guez-Lorenzo, R. de la Rica, R. A. A ´n and M. M. Stevens, Nat. Mater., 2012, 11, 604–607. Marza 7 (a) R. de la Rica and M. M. Stevens, Nat. Nanotechnol., 2012, 7, 821–824; (b) R. de la Rica and M. M. Stevens, Nat. Protoc., 2013, 8, 1759–1764; (c) D. Cecchin, R. de la Rica, R. E. S. Bain, M. W. Finnis, M. M. Stevens and G. Battaglia, Nanoscale, 2014, 6, 9559–9562. 8 Y. Lin, J. Ren and X. Qu, Acc. Chem. Res., 2014, 47, 1097–1105. 9 I. Willner, B. Shlyahovsky, M. Zayats and B. Willner, Chem. Soc. Rev., 2008, 37, 1153–1165. 10 W. Zhao, M. M. Ali, M. A. Brook and Y. Li, Angew. Chem., Int. Ed., 2008, 47, 6330–6337. 11 M. M. Ali, F. Li, Z. Zhang, K. Zhang, D.-K. Kang, J. A. Ankrum, X. C. Le and W. Zhao, Chem. Soc. Rev., 2014, 43, 3324–3341. 12 (a) L. Tang, Y. Liu, M. M. Ali, D. K. Kang, W. Zhao and J. Li, Anal. Chem., 2012, 84, 4711–4717; (b) F. Wang, C.-H. Lu, X. Liu, L. Freage and I. Willner, Anal. Chem., 2014, 86, 1614–1621. 13 H. Ma, J. Liu, M. M. Ali, M. A. I. Mahmood, L. Labanieh, M. Lu, S. M. Iqbal, Q. Zhang, W. Zhao and Y. Wan, Chem. Soc. Rev., 2015, 44, 1240–1256. 14 C. Zhu, Y. Wen, D. Li, L. Wang, S. Song, C. Fan and I. Willner, Chem. – Eur. J., 2009, 15, 11898–11903. 15 L. Wang, K. Tram, M. M. Ali, B. J. Salena, J. Li and Y. Li, Chem. – Eur. J., 2014, 20, 2420–2424. 16 A. R. Carroll, M. S. Buchanan, A. Edser, W. Hyde, M. Simpson and R. J. Quinn, J. Nat. Prod., 2004, 67, 1291–1294. 17 C. Engelbrekt, K. H. Sørensen, J. Zhang, A. C. Welinder, P. S. Jensen and J. Ulstrup, J. Mater. Chem., 2009, 19, 7839–7847.
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Cite this: DOI: 10.1039/c5cc02069g Received 12th March 2015, Accepted 23rd March 2015
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A plasmonic aptasensor for ultrasensitive detection of thrombin via arrested rolling circle amplification† Sai Wang, Sai Bi,* Zonghua Wang,* Jianfei Xia, Feifei Zhang, Min Yang, Rijun Gui, Yanhui Li and Yanzhi Xia
DOI: 10.1039/c5cc02069g www.rsc.org/chemcomm
A sensitive signal generation mechanism for gold nanoparticle growth by reducing gold ions with hydrogen peroxide is applied in a plasmonic aptasensor, achieving naked-eye detection of thrombin at the singlemolecule level based on the specific interaction of aptamer–thrombin via an arrested rolling circle amplification to yield horseradish peroxidase (HRP)-mimicking DNAzymes as biocatalysts.
Colorimetric methods have become attractive tools for biomolecule detection due to their simplicity, cost-effectiveness, and not needing sophisticated equipment.1 However, conventional colorimetric detection methods, such as the enzyme-linked immunosorbent assay (ELISA), are often based on the differentiation of two similar looking lightly colored solutions using organic compounds as substrates (e.g., 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB), 2,2 0 azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), o-phenylenediamine (OPD)), which cannot facilitate the confident detection of target molecules at low concentrations.2 Alternatively, plasmonic nanoparticle-based visual detection can potentially improve the standard of colorimetric methods,3 which are often based on target-induced aggregation of functionalized gold nanoparticles (AuNPs) with the easy observation of different colored solutions of either blue or red.4 In these assays, the spherical AuNPs had to be synthesized previously and then required specific storage and reaction conditions, especially salt concentration, to generate a colored signal.5 In addition, it is still difficult to meet the sensitivity requirements for the detection of analytes at ultralow concentrations. To address these issues, Rica and Stevens reported a plasmonic nanosensor for antigen detection down to 40 zM by means of an enzyme-guided crystal growth of gold nanostars.6 Very recently, the College of Chemical Science and Engineering, 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, P. R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: Experimental section and additional results. See DOI: 10.1039/c5cc02069g
This journal is © The Royal Society of Chemistry 2015
same group also developed a plasmonic ELISA for the detection of antigens at ultralow concentrations (1018 g mL1) with the naked eye.7 Although these plasmonic methods achieved ultrahigh sensitivity, protein enzymes, such as glucose oxidase (GOx) or catalase, were used to control the concentration of hydrogen peroxide, but these methods are often hampered by their intrinsic drawbacks, such as low operational stability, high cost of preparation and purification, sensitivity of catalytic activity to environmental conditions and difficulty of modification.8 As a promising candidate, a HRP-mimicking DNAzyme with high catalytic properties has the advantages of easy synthesis and modification with low cost, as well as an impressive stability against stringent conditions. To date, DNAzymes have been widely applied as biolabels that transduce and amplify sensing events.9 More importantly, to achieve a higher detection sensitivity, innovative amplification strategies can be incorporated into these plasmonic methods. Rolling circle amplification (RCA) is an isothermal enzymatic process to generate long single-stranded DNA or RNA using a short strand of DNA or RNA as a primer and circular DNA as a template in the presence of unique polymerases.10 Due to its simplicity and versatility, RCA has been explored extensively for the sensitive detection of various targets.11 Importantly, functional sequences (e.g., HRP-mimicking DNAzymes) can be readily incorporated into RCA products by designing the template, and can act as self reporters to generate an optical or electrochemical signal.12 In addition, aptamers are single-stranded oligonucleotides that are selected by the systematic evolution of ligands by exponential enrichment (SELEX) and have emerged as alternative biorecognition elements with a high specificity and affinity for a variety of targets ranging from metal ions and small molecules to proteins, viruses, and even cells.13 Recently, Fan’s group has found that the aptamer–thrombin complex could prevent Klenow DNA polymerase from reading through the aptameric domain, which would indirectly inhibit in vitro replication of DNA.14 Li et al. further exploited the aptamer–protein interactions that can arrest the RCA reaction using phi29 DNA polymerase with a circular DNA template.15
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Fig. 1 Native PAGE of RCA products in the presence (lane 1) and absence (lane 2) of thrombin. The concentrations of the primer, circular template and thrombin are 5 mM. M represents the marker.
Scheme 1 (a) Schematic illustration of the visual and ultrasensitive detection of thrombin using a plasmonic aptasensor based on arrested RCA. (b) Growth mechanism of AuNPs. Arrows represent the 30 -terminus of DNA strands.
Herein, we propose a plasmonic aptasensor for the naked eye detection of thrombin with ultrahigh sensitivity on the basis of arrested RCA. The principle of the strategy is presented in Scheme 1. To demonstrate the proof of principle, we chose thrombin, the central protease in the blood coagulation cascade proteases, as the target analyte of interest.16 Firstly, amino-modified DNA (1) is covalently immobilized on each well of a microtiter plate using glutaraldehyde as the coupling reagent. The coverage of DNA (1) on each well was calculated to be 9.68 pmol per well (see ESI†). A padlock DNA probe (2), consisting of five regions, is used as the circular template for the subsequent arrested RCA. Regions I and II (black, 10 mer) are complementary to the half sequence of (1); regions III and IV (blue, 17 mer) are complementary sequences to the HRP-mimicking DNAzymes; region V (purple, 15 mer) is the aptamer segment, which can bind to the fibrinogen-binding site of a-thrombin through the formation of a G-quadruplex structure with a high affinity and specificity (dissociation constant (Kd) = 25 nM).15 Therefore, the 50 - and 30 -end of the padlock probe (2) can be ligated and circularized through hybridizing regions I and II with DNA (1) in the presence of T4 DNA ligase to obtain the aptamer circular template. Subsequently, the addition of thrombin stimulates the self-assembly of region V in (2) into a tight aptamer– thrombin complex, making the circularized (2) unsuitable as a template for phi29 DNA polymerase and resulting in the prohibition of RCA. We refer to this process as arrested RCA. In contrast, in the absence of thrombin, the RCA reaction can be initiated in the presence of phi29 DNA polymerase/dNTPs by using the singlestranded DNA (1) as a primer and the circular padlock probe (2) as a template. The resultant RCA products containing multiple complementary sequences to the circle template are generated, further forming a large number of hemin/G-quadruplex HRP-mimicking DNAzymes in the presence of hemin. The reaction pathways of RCA with the aptamer circular template were verified by native polyacrylamide gel electrophoresis (PAGE) (Fig. 1). In the absence of thrombin, the aptamer circular template serves as the regular circular template for the RCA reaction in the presence of phi29 DNA polymerase/dNTPs, resulting in the generation of long strand DNA products that are too large to enter the gel by electrophoresis (lane 2). Conversely, no RCA product can be observed from the sample in the presence of
Chem. Commun.
thrombin, reliably confirming that thrombin specifically engages the aptamer into a fairly stable complex that arrests RCA by inhibiting the strand-displacement ability of phi29 DNA polymerase (lane 1). As shown in Scheme 1b, in the proposed plasmonic aptasensor we apply the hemin/G-quadruplex HRP-mimicking DNAzymes as biocatalytic labels that are linked to the growth of AuNPs to obtain blue- or red-colored solutions in the presence or absence of thrombin. In the presence of thrombin, hydrogen peroxide can reduce the gold precursor at a fast rate in the 2-(N-morpholino)ethanesulfonic acid (MES) buffer, resulting in the formation of quasi-spherical and well-dispersed AuNPs with a red color. In the absence of thrombin, the HRP-mimicking DNAzyme catalyzes the decomposition of hydrogen peroxide into H2O and O2, which slows down the kinetics of crystal growth. As a result, aggregated AuNPs with ill-defined morphology are formed, showing a blue color. Since it is easy to distinguish the two differently colored solutions (blue and red) at a glance, the plasmonic method allows ultrasensitive detection of thrombin with the naked eye. In this reaction, for the growth of AuNPs, the concentration of hydrogen peroxide is a critical condition that can affect the morphology and optical properties of AuNPs, therefore different concentrations of hydrogen peroxide were added to a 0.1 mM Au3+ solution in the presence of MES to act as a supporting reducing agent and a capping ligand.17 As shown in Fig. 2a, the tonality of the solution changes from blue to red gradually as the concentration of hydrogen peroxide increases in the range from 1 to 11 mM after reacting for 15 min. The results indicate that when the concentration of hydrogen peroxide is low, clusters of aggregated AuNPs are formed, which generates a blue color. Nevertheless, when hydrogen peroxide is at a high concentration, non-aggregated
Fig. 2 (a) Photograph and (b) UV-vis spectra showing AuNP growth after adding different concentrations of hydrogen peroxide to a growth solution containing 0.1 mM Au3+ in 1 mM MES buffer (pH 6.5). (c) UV-vis absorbance at 540 nm corresponding to different concentrations of hydrogen peroxide.
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colloidal suspensions are obtained, and the solution can be easily identified by a red color. The color change corresponds to a wavelength shift in the surface plasmon resonance of AuNPs, which was verified by UV-vis spectra (Fig. 2b). As the concentration of hydrogen peroxide decreases, the wavelength of the peak absorbance is red-shifted (see ESI,† S3), indicating the formation of aggregated AuNPs at low concentrations of hydrogen peroxide. Furthermore, AuNP growth for different concentrations of hydrogen peroxide was investigated by measuring the UV-vis absorbance at 540 nm (Fig. 2c), showing a critical point when the concentration of hydrogen peroxide is 3 mM. Overall, in this study the morphology and state of aggregation of AuNP growth is sensitive to the concentration of hydrogen peroxide as it acts as reducing agent to reduce gold ions in MES according to eqn (1) in Scheme 1b. Moreover, according to eqn (2) in Scheme 1b, the HRPmimicking DNAzyme can catalyze the decomposition of hydrogen peroxide into H2O and O2. The influences of the hemin/ G-quadruplex HRP-mimicking DNAzyme and hemin on AuNP growth were also investigated and the high catalytic activity of HRP-mimicking DNAzymes to reduce the concentration of hydrogen peroxide that directly influences the growth of AuNPs has been demonstrated (see ESI†). Subsequently, we readily adapted the sensitive detection system to biosensing strategies by using HRP-mimicking DNAzymes as labels to control the concentration of hydrogen peroxide, and further improved the naked-eye detection of the analytes at low concentrations by combining with the amplification protocol of arrested RCA as illustrated in Scheme 1. Fig. 3a shows the results for the naked-eye detection of thrombin with the proposed plasmonic aptasensor. As the concentration of thrombin increases, the red color intensifies. This observation is attributed to the decreased generation of HRP-mimicking DNAzymes via the RCA reaction as a result of the specific binding between thrombin and the aptamer in
Fig. 3 Photograph (a) and corresponding UV-vis calibration curve (b) for thrombin detection with the plasmonic aptasensor. (c and d) TEM images of AuNPs grown by the plasmonic aptasensor in the absence and presence of thrombin (1 fM), respectively. The final concentrations of hydrogen peroxide and Au3+ in the MES buffer (1 mM, pH 6.5) are 3 mM and 0.1 mM, respectively.
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circular DNA that is thus unsuitable as the template for the phi29 DNA polymerase; this increases the concentration of hydrogen peroxide and favors the generation of red solutions of AuNPs. Remarkably, the color obtained by 1017 M thrombin could be easily differentiated from the blank (thrombin concentration of 0), correspondingly achieving a limit of detection as low as B6 molecules in a 1 mL test sample. Moreover, from the calibration curve in Fig. 3b, the UV-vis absorbance at 540 nm increases with the increase in thrombin concentration, which is consistent with the color of te nanoparticle solutions observed in Fig. 3a. The ultrahigh sensitivity is attributed not only to the detection system of AuNP growth that is extremely sensitive to the concentration of hydrogen peroxide, but also to the efficient amplification of RCA and the high catalytic action of the HRPmimicking DNAzyme that has multiple turnovers through RCA. The morphology of the resultant colloidal solutions in the absence and presence of thrombin were further confirmed by TEM, and were grown as clusters of aggregated and nonaggregated AuNPs, respectively, as expected (Fig. 3c and d). Therefore, these results demonstrate the feasibility of the plasmonic aptasensor for the ultrasensitive detection of the target thrombin that controls the generation of HRP-mimicking DNAzymes via arrested RCA and further regulates the growth of AuNPs with a desired state of aggregation to yield a colored solution with a distinct tonality. Moreover, we challenged the plasmonic aptasensor with four non-cognate proteins: bovine serum albumin (BSA), bovine hemoglobin (BHb), lysozyme and nucleolin. From Fig. 4, the red color is only observed for thrombin, whereas the other samples yield blue-colored nanoparticle solutions even though the analytes are at a concentration of 1 fM. This excellent specificity is attributed to the direct concentration relationship between the HRP-mimicking DNAzyme and thrombin, based on the high selectivity and affinity of the aptamer in the circular template to thrombin for the performance of the RCA reaction. In conclusion, we have adapted the sensitive naked-eye detection system of AuNP growth through the reduction of gold ions with hydrogen peroxide as a signal generation mechanism for the fabrication of a plasmonic aptasensor. To further improve the detection sensitivity, the amplification technique of RCA is incorporated to generate a cascade of HRP-mimicking DNAzymes as biocatalysts to control the concentration of hydrogen peroxide. By rationally encoding an aptamer sequence into the circular template, an arrested RCA strategy is proposed based on the inability of phi29 DNA polymerase to read through the aptamer–thrombin complexes. Significantly, this plasmonic aptasensor achieves an ultralow detection limit of 10 aM thrombin (B6 molecules in a 1 mL test sample),
Fig. 4 Photographs for the detection of various analytes with the plasmonic aptasensor. The concentration of each analyte is 1 fM. The reaction conditions are the same as those in Fig. 3.
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which greatly improves upon the sensitivity of traditional colorimetric methods using AuNPs (ESI,† S5) and other RCA strategies for thrombin detection (ESI,† S6), giving great promise as a versatile tool for single-molecule detection in biosensing and biomedicine. This work was supported by the National Science Foundation of China (21375056, 21405086, 21475071, 21275082), the Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-12-1024), the Taishan Scholar Program of Shandong Province, the Natural Science Foundation of Shandong (ZR2014BQ001, BS2014NJ023) and the Natural Science Foundation of Qingdao (13-1-4-128-jch and 13-1-4-202-jch).
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