Analytical Biochemistry 457 (2014) 19–23
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification Cuiping Ma a,b, Wenshuo Wang a, Ashok Mulchandani b,⇑, Chao Shi a,⇑ a Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China b Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA
a r t i c l e
i n f o
Article history: Received 25 February 2014 Received in revised form 1 April 2014 Accepted 19 April 2014 Available online 26 April 2014 Keywords: Gold nanoparticle Colorimetric DNA detection Hybridization chain reaction Signal amplification Nanobiosensor
a b s t r a c t A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs. Ó 2014 Elsevier Inc. All rights reserved.
Due to their high extinction coefficient and distance-dependent optical properties, gold nanoparticles (AuNPs)1 have emerged as ideal materials for colorimetric biosensors [1–3]. The color of AuNPs is red in the dispersed state, but changes to purple or blue on aggregation because the surface plasmon band shifts to a longer wavelength [4]. As a result, AuNPs, especially DNA-functionalized AuNPs, have been successfully employed as a colorimetric probe for the detection of various analytes including nucleic acids [1,5– 9], proteins [10], metal ions [11–13], and small molecules [14,15]. The aggregation of DNA-functionalized AuNPs is usually induced by the hybridization of DNA or the increase of salt concentration [16]. Mirkin and co-workers [17] first reported the crosslinked aggregation of AuNPs induced by DNA hybridization. However, the crosslinked AuNP aggregation induced by DNA hybridization is a relatively time-consuming process owing to steric considerations and electrostatic repulsive interactions [1]. Of note, steric considerations and electrostatic repulsive interactions provided by negatively charged DNA polymers enable DNAfunctionalized AuNPs to remain stable even at relatively high salt concentrations [17]. Salt-induced aggregation is caused by the neutralization of the negative charges of DNA on AuNP surfaces ⇑ Corresponding authors. Fax: +86 84022680. E-mail addresses: [email protected] (A. Mulchandani), [email protected] (C. Shi). 1 Abbreviations used: AuNPs, gold nanoparticles; HCR, hybridization chain reaction. http://dx.doi.org/10.1016/j.ab.2014.04.022 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
[16,18]. Zhao et al. developed a simple and rapid colorimetric assay that exploited structure-switching DNA aptamers and the phenomenon of salt-induced AuNP aggregation [15]. In this assay, DNA strands attached to AuNPs were first hybridized with adenosine aptamer strands, which enhanced the stability of the AuNPs at a certain concentration of MgCl2 by providing additional negative charges. Introduction of adenosine induced the switch of aptamer structure, and dissociated the aptamer strands from the AuNPs. The dissociation of aptamers decreased the salt stability of the AuNPs, which resulted in a rapid color change from red to purple. In recent years, enzyme-free DNA circuits have attracted much attention [19]. The entropy-driven catalytic hybridization [20], triggered self-assembly [21,22], and see-saw gates [23] all can easily achieve signal amplification depending on the hybridization and strand-exchange reactions. Enzyme-free signal amplification is playing an important role in the development of biosensors and DNA nanotechnology. For instance, the hybridization chain reaction (HCR) [21,24], entropy-driven catalysis [25], and catalyzed hairpin assembly [7,26–28] have been effectively used for the design of biosensors. Moreover, DNA-based computation by strand displacement cascades has been reported [29,30]. In this work, we constructed a novel sandwich-like colorimetric system by combining DNA-functionalized AuNPs with HCR. In the system, the 30 end of the target DNA strand hybridized with the DNA strand tethered on AuNPs, and the 50 end triggered the HCR to produce a long DNA polymer. Thus, a small amount of target
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Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
DNA strands could provide substantial negative charges on AuNPs, preventing the individual red AuNPs from forming blue aggregates at relatively high salt concentrations. Using this enzyme-free and signal amplification strategy, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Materials and methods Reagents and materials All oligonucleotides (HPLC purified) used in this work (Table S1) were supplied by SBS Genetech. Co. Ltd. Gold nanoparticles with an average diameter of 15 nm were synthesized using the citrate reduction protocol reported previously [31]. The concentration of AuNPs (2.8 nM) was calculated by comparing the number of Au atoms per particle to the total number of Au atoms in the solution [32]. AuNPs were functionalized with thiol-modified oligonucleotides according to the method described in the literature [33]. The functionalized AuNPs were purified by centrifugation and removal of supernatant. Then the final particles were resuspended in sodium phosphate buffer (10 mM, 0.3 M NaCl, pH 7.5).
Amplified detection of target DNA Varied concentrations of target DNA were added to the mixture of AuNPs and hairpins H1 and H2 in different tubes. The final volume of every sample was 20 lL, and both the final concentration of H1 and that of H2 were 108 M in each sample. The mixtures were incubated at room temperature for 4 h, followed by the addition of 1 lL of 1 M MgCl2. The color changes of solutions were observed by the naked eye and photographed. Finally, the samples were diluted to 1200 lL and the 400- to 750-nm absorption spectrum of each sample was recorded with a UV/Vis spectrophotometer in 20 min. Results and discussion Design of colorimetric sensor Fig. 1 depicts the colorimetric DNA detection by enzyme-free signal amplification and gold nanoparticle. In this method, target DNA was divided into two regions (Table S1). The first region hybridized with a DNA-functionalized AuNP, and the second region as an initiator triggered a hybridization chain reaction. We used HCR to achieve signal amplification and improve sensitivity of
Gel electrophoresis of HCR products The HCR system was verified by using agarose gel electrophoresis. To ensure that H1 and H2 formed hairpin monomers, we annealed the hairpin strands by heating for 3 min at 95 °C in sodium phosphate buffer, and then allowing them to cool to room temperature. H1 and H2 were mixed in different tubes, to which different concentrations of target DNA were added. The final concentration of each hairpin—H1 and H2—was 1 lM. Hybridization chain reactions were performed at room temperature for 4 h. These reaction samples were then run on a 2% agarose gel for 30 min at 100 V and imaged under UV light. Unamplified colorimetric DNA detection We investigated the sensitivity of the colorimetric sensor without HCR with the naked eye. The DNA-functionalized AuNPs were incubated with various concentrations of target DNA (total volume 10 lL) for 10 min at room temperature, followed by the addition of 0.5 lL of 1 M MgCl2 for colorimetric detection. The color changes of solutions were observed by the naked eye, and photographed 1 min after the addition of MgCl2 (Fig. S1).
Fig.2. Gel electrophoresis of HCR products induced by different concentrations of target DNA containing 1 lM mixture of H1 and H2. Lane M, DL 2000 DNA Marker; Lanes 1–5, 3, 1, 0.3, 0.1, and 0 lM, respectively, of target DNA.
Fig.1. Schematic representation of colorimetric DNA detection by enzyme-free signal amplification and gold nanoparticle.
Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
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Fig.3. Colorimetric detection of target DNA with HCR signal amplification. Target concentration were 0, 0.5, 1, 2.5, 5, 10, 25, and 50 nM from right to left; (A) before MgCl2 addition and (B) after MgCl2 addition.
colorimetric DNA detection. The HCR system consisted of two nucleic acid hairpin species (H1 and H2) (Table S1). In the presence of target DNA, target DNA partially hybridized with the DNA probe which was immobilized on AuNPs. The initiator region of target DNA first hybridized with H1 via base-pairing to its sticky end, and subsequently opened the hairpin H1 via branch migration. The newly exposed sticky end of H1 hybridized with the sticky end of H2 and opened the hairpin to expose a sticky end on H2 that was identical in sequence to the initiator strand. Regeneration of
the initiator sequence laid the foundation for a chain reaction of alternating H1 and H2 polymerization steps leading to formation of a nicked double-stranded ‘‘polymer.’’ Thus, the resulting polymer with a large number of negative charges was linked with AuNP by target DNA. The increase of negative charges significantly enhanced the stability of AuNPs, so as to inhibit aggregation of AuNPs at an elevated salt concentration. Conversely, in the absence of the target DNA, AuNPs easily aggregated at the same salt concentration.
Fig.4. (A) The UV–visible spectrum for different concentrations of target DNA. (B) The relationship between the concentration of target DNA and A525/A700. Each value is the mean of the results of 3 experiments.
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Fig.5. Specificity of HCR-amplified detection assay. (1) Completely complementary target DNA; (2) one-base mismatched DNA; (3) two-base mismatched DNA; (4) threebase mismatched DNA.
Verification of hybridization chain reaction by gel electrophoresis In a basic HCR system, hairpin secondary structures were typically designed with stems of 18 nt and loops of 6 nt [21]. Cognizant of the fact that HCR would be slower on the surface of AuNP due to the repulsion between negatively charged AuNP and hairpin DNA strands, we designed the two hairpins with 15 nt stems and 6 nt loops to accelerate the rate of HCR on AuNPs because the shortened stems facilitated the strand exchange processes of hairpins. To confirm the formation of long DNA polymerization products in the HCR, the progress of HCR was visualized by gel electrophoresis. As shown in Fig. 2, HCRs were triggered by different amounts of target DNA strands. Of note, as seen in the electrophoresis gel, a small amount of long polymerization products was formed in the absence of target DNA. However, we hypothesize that these polymerization products could not be attached to AuNPs and therefore would be ineffective in preventing AuNPs aggregation at a higher salt concentration, which was validated experimentally (Fig. 3). Analytical performance of the colorimetric sensor The sensitivity of the AuNP-based colorimetric sensor without HCR was examined initially. The results indicated that target DNA at concentrations as low as 10 nM could be rapidly detected with the naked eye (Fig. S1). To improve the sensitivity of this approach, we used HCR to achieve signal amplification. Then, we investigated the effect of combining HCR with the above colorimetric assay by introducing H1 and H2 hairpins. Fig. 3 shows the degree of red color formation as a function of target DNA concentration before and after MgCl2 addition in the HCR signal-amplified colorimetric assay. As seen in the figure, a slight red color was visually observable beginning from 0.5 nM target DNA and became intense at higher DNA concentrations. This sensitivity was better than the 10 nM DNA determined in the unamplified colorimetric assay described above. The improved sensitivity is attributed to the large amounts of negative charges and high steric stabilization of long HCR polymers that prevented AuNPs from coming closer to form aggregates. To further quantify the target concentration, the ratios of the absorbance at 525 and 700 nm were plotted as a function of DNA concentration (Fig. 4). The value of A525/A700 increased with increasing target concentration. The detection limit of this method was estimated to be 0.5 nM. We found that the sensitivity was
remarkably improved as compared to the classic AuNP-based colorimetric method by at least 20-fold [34]. Selectivity of the colorimetric sensor The sequence specificity of this method was evaluated by testing the response of the assay to targets with one, two, and three mismatches. As shown in Fig. 5, the A525/A700 ratios for the three mismatched DNAs were obviously lower compared to fully complimentary target, which confirmed that it was easy to distinguish the complementary target from the mismatched DNA by the newly developed assay. These results indicated the high specificity of our method. Conclusions We constructed an enzyme-free, isothermal signal amplification method for colorimetric detection of target DNA. This method is simple, sensitive, and selective, allowing detection of as low as 0.5 nM target DNA with the naked eye. Compared with the unmodified AuNP-based colorimetric DNA detection method [35], our approach, which only responds to reaction products triggered by target DNA, is hardly affected by nonspecific products or nontarget single-strand DNA. Our method also has great potential for the detection of other analytes on the condition that the target analytes could make HCR products attach to AuNPs. Furthermore, it is possible to develop new functional nanomaterials by combining nanoscale HCR products with AuNPs. Acknowledgments The work was supported by National Natural Science Foundation of China (31170758, 21375071, 21307064), Science and Technology Development Project of Shandong Province (2012GSF12001), Public Sphere Support Program of Qingdao (12-1-3-62-nsh), and The Natural Science Foundation of Shandong Province (ZR2011BQ023). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.022.
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Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification Cuiping Ma a,b, Wenshuo Wang a, Ashok Mulchandani b,⇑, Chao Shi a,⇑ a Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China b Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA
a r t i c l e
i n f o
Article history: Received 25 February 2014 Received in revised form 1 April 2014 Accepted 19 April 2014 Available online 26 April 2014 Keywords: Gold nanoparticle Colorimetric DNA detection Hybridization chain reaction Signal amplification Nanobiosensor
a b s t r a c t A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs. Ó 2014 Elsevier Inc. All rights reserved.
Due to their high extinction coefficient and distance-dependent optical properties, gold nanoparticles (AuNPs)1 have emerged as ideal materials for colorimetric biosensors [1–3]. The color of AuNPs is red in the dispersed state, but changes to purple or blue on aggregation because the surface plasmon band shifts to a longer wavelength [4]. As a result, AuNPs, especially DNA-functionalized AuNPs, have been successfully employed as a colorimetric probe for the detection of various analytes including nucleic acids [1,5– 9], proteins [10], metal ions [11–13], and small molecules [14,15]. The aggregation of DNA-functionalized AuNPs is usually induced by the hybridization of DNA or the increase of salt concentration [16]. Mirkin and co-workers [17] first reported the crosslinked aggregation of AuNPs induced by DNA hybridization. However, the crosslinked AuNP aggregation induced by DNA hybridization is a relatively time-consuming process owing to steric considerations and electrostatic repulsive interactions [1]. Of note, steric considerations and electrostatic repulsive interactions provided by negatively charged DNA polymers enable DNAfunctionalized AuNPs to remain stable even at relatively high salt concentrations [17]. Salt-induced aggregation is caused by the neutralization of the negative charges of DNA on AuNP surfaces ⇑ Corresponding authors. Fax: +86 84022680. E-mail addresses: [email protected] (A. Mulchandani), [email protected] (C. Shi). 1 Abbreviations used: AuNPs, gold nanoparticles; HCR, hybridization chain reaction. http://dx.doi.org/10.1016/j.ab.2014.04.022 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
[16,18]. Zhao et al. developed a simple and rapid colorimetric assay that exploited structure-switching DNA aptamers and the phenomenon of salt-induced AuNP aggregation [15]. In this assay, DNA strands attached to AuNPs were first hybridized with adenosine aptamer strands, which enhanced the stability of the AuNPs at a certain concentration of MgCl2 by providing additional negative charges. Introduction of adenosine induced the switch of aptamer structure, and dissociated the aptamer strands from the AuNPs. The dissociation of aptamers decreased the salt stability of the AuNPs, which resulted in a rapid color change from red to purple. In recent years, enzyme-free DNA circuits have attracted much attention [19]. The entropy-driven catalytic hybridization [20], triggered self-assembly [21,22], and see-saw gates [23] all can easily achieve signal amplification depending on the hybridization and strand-exchange reactions. Enzyme-free signal amplification is playing an important role in the development of biosensors and DNA nanotechnology. For instance, the hybridization chain reaction (HCR) [21,24], entropy-driven catalysis [25], and catalyzed hairpin assembly [7,26–28] have been effectively used for the design of biosensors. Moreover, DNA-based computation by strand displacement cascades has been reported [29,30]. In this work, we constructed a novel sandwich-like colorimetric system by combining DNA-functionalized AuNPs with HCR. In the system, the 30 end of the target DNA strand hybridized with the DNA strand tethered on AuNPs, and the 50 end triggered the HCR to produce a long DNA polymer. Thus, a small amount of target
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Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
DNA strands could provide substantial negative charges on AuNPs, preventing the individual red AuNPs from forming blue aggregates at relatively high salt concentrations. Using this enzyme-free and signal amplification strategy, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Materials and methods Reagents and materials All oligonucleotides (HPLC purified) used in this work (Table S1) were supplied by SBS Genetech. Co. Ltd. Gold nanoparticles with an average diameter of 15 nm were synthesized using the citrate reduction protocol reported previously [31]. The concentration of AuNPs (2.8 nM) was calculated by comparing the number of Au atoms per particle to the total number of Au atoms in the solution [32]. AuNPs were functionalized with thiol-modified oligonucleotides according to the method described in the literature [33]. The functionalized AuNPs were purified by centrifugation and removal of supernatant. Then the final particles were resuspended in sodium phosphate buffer (10 mM, 0.3 M NaCl, pH 7.5).
Amplified detection of target DNA Varied concentrations of target DNA were added to the mixture of AuNPs and hairpins H1 and H2 in different tubes. The final volume of every sample was 20 lL, and both the final concentration of H1 and that of H2 were 108 M in each sample. The mixtures were incubated at room temperature for 4 h, followed by the addition of 1 lL of 1 M MgCl2. The color changes of solutions were observed by the naked eye and photographed. Finally, the samples were diluted to 1200 lL and the 400- to 750-nm absorption spectrum of each sample was recorded with a UV/Vis spectrophotometer in 20 min. Results and discussion Design of colorimetric sensor Fig. 1 depicts the colorimetric DNA detection by enzyme-free signal amplification and gold nanoparticle. In this method, target DNA was divided into two regions (Table S1). The first region hybridized with a DNA-functionalized AuNP, and the second region as an initiator triggered a hybridization chain reaction. We used HCR to achieve signal amplification and improve sensitivity of
Gel electrophoresis of HCR products The HCR system was verified by using agarose gel electrophoresis. To ensure that H1 and H2 formed hairpin monomers, we annealed the hairpin strands by heating for 3 min at 95 °C in sodium phosphate buffer, and then allowing them to cool to room temperature. H1 and H2 were mixed in different tubes, to which different concentrations of target DNA were added. The final concentration of each hairpin—H1 and H2—was 1 lM. Hybridization chain reactions were performed at room temperature for 4 h. These reaction samples were then run on a 2% agarose gel for 30 min at 100 V and imaged under UV light. Unamplified colorimetric DNA detection We investigated the sensitivity of the colorimetric sensor without HCR with the naked eye. The DNA-functionalized AuNPs were incubated with various concentrations of target DNA (total volume 10 lL) for 10 min at room temperature, followed by the addition of 0.5 lL of 1 M MgCl2 for colorimetric detection. The color changes of solutions were observed by the naked eye, and photographed 1 min after the addition of MgCl2 (Fig. S1).
Fig.2. Gel electrophoresis of HCR products induced by different concentrations of target DNA containing 1 lM mixture of H1 and H2. Lane M, DL 2000 DNA Marker; Lanes 1–5, 3, 1, 0.3, 0.1, and 0 lM, respectively, of target DNA.
Fig.1. Schematic representation of colorimetric DNA detection by enzyme-free signal amplification and gold nanoparticle.
Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
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Fig.3. Colorimetric detection of target DNA with HCR signal amplification. Target concentration were 0, 0.5, 1, 2.5, 5, 10, 25, and 50 nM from right to left; (A) before MgCl2 addition and (B) after MgCl2 addition.
colorimetric DNA detection. The HCR system consisted of two nucleic acid hairpin species (H1 and H2) (Table S1). In the presence of target DNA, target DNA partially hybridized with the DNA probe which was immobilized on AuNPs. The initiator region of target DNA first hybridized with H1 via base-pairing to its sticky end, and subsequently opened the hairpin H1 via branch migration. The newly exposed sticky end of H1 hybridized with the sticky end of H2 and opened the hairpin to expose a sticky end on H2 that was identical in sequence to the initiator strand. Regeneration of
the initiator sequence laid the foundation for a chain reaction of alternating H1 and H2 polymerization steps leading to formation of a nicked double-stranded ‘‘polymer.’’ Thus, the resulting polymer with a large number of negative charges was linked with AuNP by target DNA. The increase of negative charges significantly enhanced the stability of AuNPs, so as to inhibit aggregation of AuNPs at an elevated salt concentration. Conversely, in the absence of the target DNA, AuNPs easily aggregated at the same salt concentration.
Fig.4. (A) The UV–visible spectrum for different concentrations of target DNA. (B) The relationship between the concentration of target DNA and A525/A700. Each value is the mean of the results of 3 experiments.
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Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
Fig.5. Specificity of HCR-amplified detection assay. (1) Completely complementary target DNA; (2) one-base mismatched DNA; (3) two-base mismatched DNA; (4) threebase mismatched DNA.
Verification of hybridization chain reaction by gel electrophoresis In a basic HCR system, hairpin secondary structures were typically designed with stems of 18 nt and loops of 6 nt [21]. Cognizant of the fact that HCR would be slower on the surface of AuNP due to the repulsion between negatively charged AuNP and hairpin DNA strands, we designed the two hairpins with 15 nt stems and 6 nt loops to accelerate the rate of HCR on AuNPs because the shortened stems facilitated the strand exchange processes of hairpins. To confirm the formation of long DNA polymerization products in the HCR, the progress of HCR was visualized by gel electrophoresis. As shown in Fig. 2, HCRs were triggered by different amounts of target DNA strands. Of note, as seen in the electrophoresis gel, a small amount of long polymerization products was formed in the absence of target DNA. However, we hypothesize that these polymerization products could not be attached to AuNPs and therefore would be ineffective in preventing AuNPs aggregation at a higher salt concentration, which was validated experimentally (Fig. 3). Analytical performance of the colorimetric sensor The sensitivity of the AuNP-based colorimetric sensor without HCR was examined initially. The results indicated that target DNA at concentrations as low as 10 nM could be rapidly detected with the naked eye (Fig. S1). To improve the sensitivity of this approach, we used HCR to achieve signal amplification. Then, we investigated the effect of combining HCR with the above colorimetric assay by introducing H1 and H2 hairpins. Fig. 3 shows the degree of red color formation as a function of target DNA concentration before and after MgCl2 addition in the HCR signal-amplified colorimetric assay. As seen in the figure, a slight red color was visually observable beginning from 0.5 nM target DNA and became intense at higher DNA concentrations. This sensitivity was better than the 10 nM DNA determined in the unamplified colorimetric assay described above. The improved sensitivity is attributed to the large amounts of negative charges and high steric stabilization of long HCR polymers that prevented AuNPs from coming closer to form aggregates. To further quantify the target concentration, the ratios of the absorbance at 525 and 700 nm were plotted as a function of DNA concentration (Fig. 4). The value of A525/A700 increased with increasing target concentration. The detection limit of this method was estimated to be 0.5 nM. We found that the sensitivity was
remarkably improved as compared to the classic AuNP-based colorimetric method by at least 20-fold [34]. Selectivity of the colorimetric sensor The sequence specificity of this method was evaluated by testing the response of the assay to targets with one, two, and three mismatches. As shown in Fig. 5, the A525/A700 ratios for the three mismatched DNAs were obviously lower compared to fully complimentary target, which confirmed that it was easy to distinguish the complementary target from the mismatched DNA by the newly developed assay. These results indicated the high specificity of our method. Conclusions We constructed an enzyme-free, isothermal signal amplification method for colorimetric detection of target DNA. This method is simple, sensitive, and selective, allowing detection of as low as 0.5 nM target DNA with the naked eye. Compared with the unmodified AuNP-based colorimetric DNA detection method [35], our approach, which only responds to reaction products triggered by target DNA, is hardly affected by nonspecific products or nontarget single-strand DNA. Our method also has great potential for the detection of other analytes on the condition that the target analytes could make HCR products attach to AuNPs. Furthermore, it is possible to develop new functional nanomaterials by combining nanoscale HCR products with AuNPs. Acknowledgments The work was supported by National Natural Science Foundation of China (31170758, 21375071, 21307064), Science and Technology Development Project of Shandong Province (2012GSF12001), Public Sphere Support Program of Qingdao (12-1-3-62-nsh), and The Natural Science Foundation of Shandong Province (ZR2011BQ023). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.04.022.
Colorimetric DNA detection by hybridization chain reaction for signal amplification / C. Ma et al. / Anal. Biochem. 457 (2014) 19–23
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