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Hairpin DNA probes based on target-induced in situ generation of luminescent silver nanoclusters†
Received 13th February 2014, Accepted 25th March 2014
Yan Xiao,a Zhengjun Wu,a Kwok-Yin Wongb and Zhihong Liu*a
DOI: 10.1039/c4cc01154f www.rsc.org/chemcomm
Novel hairpin DNA probes are designed and constructed based on target-induced in situ generation of luminescent silver nanoclusters. This design allows specific and versatile detection of diverse targets with easy operation and low cost.
Nucleic acids have been widely recognized as ideal candidates for engineering molecular probes because of their unique molecule recognition capabilities.1 There has been an increasing trend to use specifically designed nucleic acid probes in biosensing in recent years.2 Under most circumstances, nucleic acid probes require pre-labeling of a signal source, which needs considerable timeconsumption and may suffer from high background.3 Besides, the existence of an external label may impact the interaction of probes with targets. Some label-free nucleic acid probes utilizing environment-sensitive luminescent molecules, such as intercalating organic dyes or metal complexes, are able to overcome the above drawbacks,4 yet they are subject to potential false positive responses due to nonspecific adsorption of the luminescent molecules to biomolecules. In order to compensate or eliminate this, complex structures such as multiple secondary structure elements are adopted, accompanying with increased cost. Undoubtedly, a simple and effective configuration is an important goal in molecular probe engineering and is still desired for fabrication of nucleic acid probes. To this end, we herein report on the design and utilization of novel hairpin DNA probes, simply comprising an ssDNA chain without any label or complex construction. It takes the advantage of the in situ generation of ultrasmall luminescent silver nanoclusters (Ag NCs), which, as previously revealed, can readily grow on a specific ssDNA scaffold under mild conditions with sequence-dependence.5 The basic principle in the probe
design is based on the target-triggered release of the ssDNA scaffold which then acts as the template for the subsequent growth of Ag NCs in situ. To combine the unique recognition capability of nucleic acids with in situ synthesis of Ag NCs and to obtain a targetinduced turn-on signal, the probe was designed to contain three segments (Scheme 1A): a segment for formation of Ag NCs, i.e., the nucleation sequence (green and blue part), a target recognition segment (black part) and a blocking segment (purple part). In the normal state, the blocking segment hybridizes with the nucleation sequence and thus locks the probe, so that the growth of Ag NCs on the template is prohibited. When a target is introduced, the specific combination of the target with the recognition sequence releases the Ag NC nucleation sequence, where fluorescent Ag NCs can be generated providing readout signal for the sensing of the target. This design is relatively simple and straightforward. Differing from normal nucleic acid probes with tagged signaling species (fluorophores), this design could avoid environmental alterations of the fluorophores in target sensing processes and maintain the binding affinity and specificity of the probe. In another aspect, it is also distinguished from those environment-sensitive fluorophorebased label-free probes, because the strict sequence-dependence
a
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected]; Fax: +86 27-6875-4067 b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hunghom, Kowloon, Hong Kong, China † Electronic supplementary information (ESI) available: Experimental details, DNA sequences, the characterization of Ag nanoclusters and control experiments. See DOI: 10.1039/c4cc01154f
This journal is © The Royal Society of Chemistry 2014
Scheme 1 (A) Schematic illustration of the structure of the hairpin DNA probe. (B) Schematic illustration of the analysis of the HBV gene using the hairpin DNA probe.
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of Ag NC synthesis precludes possible false-positive responses and hence increases the robustness of sensing. Previous studies have shown that ultrasmall fluorescent Ag NCs can be produced by chemical reduction of silver ions under mild conditions on the cytosine-rich oligonucleotide.6 We hereby chose a typical cytosine-rich sequence containing 12 bases (Table S1, DNA-a, ESI†) as the template for the generation of Ag NCs.7 As a proof-of-concept demonstration, the hepatitis B virus (HBV) surfaceantigen gene8 (Table S1, target 1 with 30-base sequence, ESI†) was chosen as the target analyte. As shown in Scheme 1B, a DNA sequence complementary to the HBV gene was used as the target recognition segment, and a 7-base sequence complementary to the green part of the Ag NC template as the blocking segment (Table S1, defined as probe 1, ESI†). We first assessed the feasibility of our design by investigating the generation of Ag NCs in different situations. When using the 12-base nucleation sequence alone as the template, Ag NCs with light absorption at 440 and 580 nm and photoluminescence at 655 nm were produced through the reduction of AgNO3 with NaHB4 (Fig. S1, ESI†). The absorption at 580 nm is assigned to the characteristic peak of ultrasmall Ag clusters,5 while the absorption at 440 nm originates from the plasmon resonance of larger Ag nanoparticles which has no sequence specificity. When the reduction of AgNO3 was conducted with the probe in its normal state, in which the blocking segment hybridized with parts of the nucleation sequence to form a hairpin structure, no characteristic absorption of Ag NCs was observed (curve a in Fig. 1A). Meanwhile, the photoluminescence spectra exhibited only very weak signals (Fig. 1B, curves a and b), which can be attributed to the trace amount of free template due to the incomplete hybridization. Since the 12-base nucleation sequence was only partly hybridized with the blocking segment, we further checked whether the remaining part overhanging at the end of the hairpin stem (Scheme 1A, the blue part) could afford the growth of Ag NCs. When using the uncaged 5-base sequence (Table S1, DNA-b, ESI†) as a scaffold for Ag NC synthesis, no optical signal of Ag NCs was detected (Fig. S2, ESI†). To the locked probe solution, we then introduced the target HBV gene, which hybridizes with the recognition segment at the loop region of the probe, followed by conducting the reduction of AgNO3. The photophysical signals of Ag NCs recorded under such a situation are shown in Fig. 1A (curve b) and Fig. 1B (curves c and d).
Fig. 1 (A) UV-vis absorption spectra of the silver species synthesized with the probe (a), the probe–HBV gene duplex (b) and the HBV gene (c) as the scaffold, respectively. (B) Fluorescence excitation and emission spectra of silver species produced in different situations (a and b: probe; c and d: probe–HBV gene duplex; e and f: HBV gene).
4850 | Chem. Commun., 2014, 50, 4849--4852
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Note that the Ag NCs thus produced showed a slight shift in the characteristic absorption and emission maxima as compared with the 12-base template alone. This phenomenon is commonly observed in DNA-templated Ag NC synthesis and can be attributed to the change in the microenvironment of the DNA-Ag NCs.9 The generation of Ag NCs was also evidenced by the TEM image which clearly showed the existence of small particles with an average size of 1.88 0.25 nm (Fig. S3, ESI†). To exclude possible contribution from the HBV gene to the formation of Ag NCs, this single-strand DNA was also used as a template to confirm that no Ag NCs were produced (curve c in Fig. 1A and curves e and f in Fig. 1B), which is also evidence for the sequence-dependence of Ag NC growth. On the other hand, we also examined whether the growth of Ag NCs on the DNA scaffold would affect the stability of the already formed DNA duplex (recognition sequence-HBV gene) by polyacrylamide gel electrophoresis analysis (Fig. S4, ESI†). After the synthesis of Ag NCs on the formed duplex, the gel band was similar to the duplex without Ag NCs and no obvious band for ssDNA was observed, suggesting that the hybridized structure was not affected by the chemical reduction process. The above results indicated the effective locking of the nucleation sequence by the blocking segment as well as the successful release of the template upon the reaction of the target with the probe. Following the verification of the ability of this hairpin probe to control the in situ growth of Ag NCs, we further optimized the length of the blocking sequence. According to the principle of the probe design, the length of the blocking sequence can be a key factor to decide the signal-to-background ratio. A longer blocking sequence is able to hybridize with the nucleation sequence better and lock the probe more tightly, so that the portion of unlocked probe, i.e., the free template of Ag NCs will be reduced leading to a lowered background. On the other hand, however, the longer blocking sequence may hinder the unlocking of the probe, which will impair the release of the template and thus leads to a lowered signal in the target sensing process. Therefore, the length of the blocking sequence needs to be rationalized to balance between the two contrary effects. To this end, we further designed two probes (Table S1, probe 2 and 3, ESI†) containing the same recognition sequence and nucleation sequence as probe 1, while the length of the blocking sequence of probe 2 and 3 was 5 and 10 bases, respectively. Upon using the locked probes as templates to synthesize Ag NCs, the product of probe 2 with a 5-base blocking sequence exhibited strong absorption of Ag NCs, while the absorption of the other two probes was very weak (Fig. S5A, ESI†). The comparison of the fluorescence emission of these three systems also showed the same results (Fig. S5B, ESI†). It is concluded that considerable unlocked templates existed in the system of probe 2 due to the incomplete hybridization. It is also seen that the background of probe 1 (7-base) and probe 3 (10-base) did not differ too much, indicating that the hybridization between 7 pairs of nucleobases was basically adequate for the locking of the probe. The Ag NCs were synthesized by adding the same amount of target DNA to the three probes (Fig. 2). For probe 2, the Ag NC emission did not show much enhancement in the presence of
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Fig. 2 The fluorescence emission of Ag NCs produced on HBV gene probes (250 nM) with varying lengths (5, 7 and 10 bases) of blocking sequence. The black bars are the probes in their locking state as the template for Ag NC synthesis, while the red bars are the situations of probes in the presence of the same concentration (250 nM) of targets.
target DNA. Whilst the fluorescence of probe 3, as speculated, was much lower than the other two. This can be explained by the over strong hybridization between 10 base pairs which inhibited the release of the Ag NC template. In terms of the signal-to-background ratio which decides the sensitivity of assays, probe 1 with the 7-base blocking sequence afforded the best efficiency. As the cytosine-rich sequence may have an effect on the signal, we also designed two other target DNAs (Table S1, target 2 and 3 with 25 and 20 bases, respectively, ESI†) to optimize the target DNA sequence length. As shown in Fig. S6 (ESI†), decreasing the target sequence length resulted in a weakened signal and the optimal signal was obtained using the 30-base sequence as the target. We then investigated the performance of the probe in the detection of the HBV gene (target 1), using the probe with a 7-base blocking sequence. With the introduction of increasing amounts of HBV gene into the probe solution, the fluorescence emission intensity at 600 nm increased gradually (Fig. 3A), which, as expected, was a result of the formation of Ag NCs on the released nucleation sequence. The relative fluorescence intensity of Ag NCs ((F – F0)/F0, in which F and F0 represent the emission intensity of Ag NCs in the presence and in the absence of target molecules, respectively) was dependent on the concentration of HBV gene, and a linear calibration was obtained within 10–200 nM (Fig. 3B). The limit of detection, at a signal-to-noise ratio of 3.0,
Fig. 3 (A) The fluorescence emission of DNA–Ag NCs in the presence of varying concentrations (0, 10, 20, 40, 60, 80, 100, 120, 150, 200 nM) of HBV gene. (B) The linear relationship between the relative fluorescence intensity (F F0)/F0 and the concentration of HBV gene ranging from 10 to 200 nM.
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was estimated to be 3.0 nM. To evaluate the sequence specificity of this probe, we also investigated the response of the probe towards two other sequences, a single-base mismatched (SBM) sequence and a non-complementary (NC) sequence. From the results shown in Fig. S7 (ESI†), we could conclude that the Ag NC-based hairpin probe has the capability to distinguish target DNA from NC and SBM DNAs. We also detected the recovery of the HBV gene in spiked human serum samples. Three concentrations of the target (40, 80 and 120 nM) were spiked into 50-fold diluted human serum. The recovery values were in the range of 96–103% (Table S2, ESI†), indicating that the probe is capable of analysing real biological samples without interference. Thus far, we have demonstrated the feasibility of this novel hairpin DNA probe and validated its applicability in DNA sensing. In order to find out whether interactions other than the hybridization of oligonucleotides, such as targets that can alter the conformation of the probe, can also give out a sensing signal, another three-segment probe for the detection of the protein thrombin was constructed. The similar nucleation sequence and blocking sequence were used to form the hairpin probe (Table S1, ESI†), but a sequence of thrombin aptamer was embedded in the loop region (Fig. S8, ESI†). Considering the relatively large size of thrombin protein which might cause a steric hindrance preventing the formation of Ag NCs, we inserted a spacer containing nine T bases between the sequence of the Ag NC nucleation sequence and the thrombin aptamer. When the target protein was added to the probe and reacted with the aptamer, the conformation of the latter was changed leading to the unlocking of the probe. As a result, luminescent Ag NCs were generated via the reduction of silver ions directly in the solution. The Ag NCs produced in this case showed a characteristic absorption at 600 nm and a maximal fluorescence emission at 660 nm, respectively (Fig. S9, ESI†). The shifts in spectral maxima can be explained by the change in the microenvironment of the DNA–Ag NCs as mentioned above. Similarly, the introduction of thrombin resulted in the increase of the fluorescence emission at 660 nm (Fig. S10A, ESI†). The fluorescence intensity enhancement of the DNA–Ag NCs was dependent on the amount of thrombin introduced (Fig. S10B, ESI†). In a control experiment where some other species including proteins and amino acids were added, even with a concentration 30 times higher than that of thrombin, the emission at 660 nm showed no obvious change (Fig. S11, ESI†), which precluded possible influence of these species on the fluorescence emission and confirmed that the turn-on signal was a result of the formation of Ag NCs induced exclusively by the target. In summary, we have developed a novel hairpin DNA probe, which simply consists of three DNA sequences: a nucleation sequence as the template for Ag NC formation, a recognition sequence for specific targets and a short blocking sequence. The probe is based on target-induced release of the Ag NC template and subsequent in situ generation of Ag NCs. The designed probe is a simple single-stranded DNA without any modification or complex structure. By embedding different recognition sequences, the probes can be conveniently used for specific and versatile detection of diverse targets. This novel design may provide a simple and flexible strategy for nucleic acid-based molecular probe engineering.
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This work was supported by the National Natural Science Foundation of China (No. 21075094, 21375098), the National Basic Research of China (973 program, No. 2011CB933600) and the Program for New Century Excellent Talents in University (NCET-11-0402).
Published on 25 March 2014. Downloaded by Universiteit Utrecht on 22/10/2014 06:22:41.
Notes and references 1 (a) C. H. Lu, B. Willner and I. Willner, ACS Nano, 2013, 7, 8320; (b) O. I. Wilner and I. Willner, Chem. Rev., 2012, 112, 2528. 2 (a) H. Zhang, F. Li, B. Dever, X. F. Li and X. C. Le, Chem. Rev., 2013, 113, 2812; (b) X. Su, X. Xiao, C. Zhang and M. Zhao, Appl. Spectrosc., 2012, 66, 1249. 3 (a) H. Xu and M. Hepel, Anal. Chem., 2011, 83, 813; (b) Y. Guo, H. Wang, Y. Sun and B. Qu, Chem. Commun., 2012, 48, 3221.
4852 | Chem. Commun., 2014, 50, 4849--4852
ChemComm 4 (a) B. Li, H. Wei and S. Dong, Chem. Commun., 2007, 73; (b) H. Z. He, D. S. H. Chan, C. H. Leung and D. L. Ma, Chem. Commun., 2012, 48, 9462. 5 (a) J. T. Petty, J. Zheng, N. V. Hud and R. M. Dickson, J. Am. Chem. Soc., 2004, 126, 5207; (b) Y. Xiao, F. Shu, K. Y. Wong and Z. Liu, Anal. Chem., 2013, 85, 8493. 6 C. M. Ritchie, K. R. Johnsen, J. R. Kiser, Y. Antoku, R. M. Dickson and J. T. Petty, J. Phys. Chem. C, 2007, 111, 175. 7 (a) C. I. Richards, S. Choi, J. C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y. L. Tzeng and R. M. Dickson, J. Am. Chem. Soc., 2008, 130, 5038; (b) J. Li, X. Zhong, H. Zhang, X. C. Le and J. J. Zhu, Anal. Chem., 2012, 84, 5170. 8 S. I. Stoeva, J. S. Lee, C. S. Thaxton and C. A. Mirkin, Angew. Chem., Int. Ed., 2006, 45, 3303. 9 (a) L. Zhang, J. Zhu, S. Guo, T. Li, J. Li and E. Wang, J. Am. Chem. Soc., 2013, 135, 2303; (b) J. Sharma, H. C. Yeh, H. Yoo, J. H. Werner and J. S. Martinez, Chem. Commun., 2010, 46, 3280.
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Cite this: Chem. Commun., 2014, 50, 4849
Hairpin DNA probes based on target-induced in situ generation of luminescent silver nanoclusters†
Received 13th February 2014, Accepted 25th March 2014
Yan Xiao,a Zhengjun Wu,a Kwok-Yin Wongb and Zhihong Liu*a
DOI: 10.1039/c4cc01154f www.rsc.org/chemcomm
Novel hairpin DNA probes are designed and constructed based on target-induced in situ generation of luminescent silver nanoclusters. This design allows specific and versatile detection of diverse targets with easy operation and low cost.
Nucleic acids have been widely recognized as ideal candidates for engineering molecular probes because of their unique molecule recognition capabilities.1 There has been an increasing trend to use specifically designed nucleic acid probes in biosensing in recent years.2 Under most circumstances, nucleic acid probes require pre-labeling of a signal source, which needs considerable timeconsumption and may suffer from high background.3 Besides, the existence of an external label may impact the interaction of probes with targets. Some label-free nucleic acid probes utilizing environment-sensitive luminescent molecules, such as intercalating organic dyes or metal complexes, are able to overcome the above drawbacks,4 yet they are subject to potential false positive responses due to nonspecific adsorption of the luminescent molecules to biomolecules. In order to compensate or eliminate this, complex structures such as multiple secondary structure elements are adopted, accompanying with increased cost. Undoubtedly, a simple and effective configuration is an important goal in molecular probe engineering and is still desired for fabrication of nucleic acid probes. To this end, we herein report on the design and utilization of novel hairpin DNA probes, simply comprising an ssDNA chain without any label or complex construction. It takes the advantage of the in situ generation of ultrasmall luminescent silver nanoclusters (Ag NCs), which, as previously revealed, can readily grow on a specific ssDNA scaffold under mild conditions with sequence-dependence.5 The basic principle in the probe
design is based on the target-triggered release of the ssDNA scaffold which then acts as the template for the subsequent growth of Ag NCs in situ. To combine the unique recognition capability of nucleic acids with in situ synthesis of Ag NCs and to obtain a targetinduced turn-on signal, the probe was designed to contain three segments (Scheme 1A): a segment for formation of Ag NCs, i.e., the nucleation sequence (green and blue part), a target recognition segment (black part) and a blocking segment (purple part). In the normal state, the blocking segment hybridizes with the nucleation sequence and thus locks the probe, so that the growth of Ag NCs on the template is prohibited. When a target is introduced, the specific combination of the target with the recognition sequence releases the Ag NC nucleation sequence, where fluorescent Ag NCs can be generated providing readout signal for the sensing of the target. This design is relatively simple and straightforward. Differing from normal nucleic acid probes with tagged signaling species (fluorophores), this design could avoid environmental alterations of the fluorophores in target sensing processes and maintain the binding affinity and specificity of the probe. In another aspect, it is also distinguished from those environment-sensitive fluorophorebased label-free probes, because the strict sequence-dependence
a
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected]; Fax: +86 27-6875-4067 b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hunghom, Kowloon, Hong Kong, China † Electronic supplementary information (ESI) available: Experimental details, DNA sequences, the characterization of Ag nanoclusters and control experiments. See DOI: 10.1039/c4cc01154f
This journal is © The Royal Society of Chemistry 2014
Scheme 1 (A) Schematic illustration of the structure of the hairpin DNA probe. (B) Schematic illustration of the analysis of the HBV gene using the hairpin DNA probe.
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of Ag NC synthesis precludes possible false-positive responses and hence increases the robustness of sensing. Previous studies have shown that ultrasmall fluorescent Ag NCs can be produced by chemical reduction of silver ions under mild conditions on the cytosine-rich oligonucleotide.6 We hereby chose a typical cytosine-rich sequence containing 12 bases (Table S1, DNA-a, ESI†) as the template for the generation of Ag NCs.7 As a proof-of-concept demonstration, the hepatitis B virus (HBV) surfaceantigen gene8 (Table S1, target 1 with 30-base sequence, ESI†) was chosen as the target analyte. As shown in Scheme 1B, a DNA sequence complementary to the HBV gene was used as the target recognition segment, and a 7-base sequence complementary to the green part of the Ag NC template as the blocking segment (Table S1, defined as probe 1, ESI†). We first assessed the feasibility of our design by investigating the generation of Ag NCs in different situations. When using the 12-base nucleation sequence alone as the template, Ag NCs with light absorption at 440 and 580 nm and photoluminescence at 655 nm were produced through the reduction of AgNO3 with NaHB4 (Fig. S1, ESI†). The absorption at 580 nm is assigned to the characteristic peak of ultrasmall Ag clusters,5 while the absorption at 440 nm originates from the plasmon resonance of larger Ag nanoparticles which has no sequence specificity. When the reduction of AgNO3 was conducted with the probe in its normal state, in which the blocking segment hybridized with parts of the nucleation sequence to form a hairpin structure, no characteristic absorption of Ag NCs was observed (curve a in Fig. 1A). Meanwhile, the photoluminescence spectra exhibited only very weak signals (Fig. 1B, curves a and b), which can be attributed to the trace amount of free template due to the incomplete hybridization. Since the 12-base nucleation sequence was only partly hybridized with the blocking segment, we further checked whether the remaining part overhanging at the end of the hairpin stem (Scheme 1A, the blue part) could afford the growth of Ag NCs. When using the uncaged 5-base sequence (Table S1, DNA-b, ESI†) as a scaffold for Ag NC synthesis, no optical signal of Ag NCs was detected (Fig. S2, ESI†). To the locked probe solution, we then introduced the target HBV gene, which hybridizes with the recognition segment at the loop region of the probe, followed by conducting the reduction of AgNO3. The photophysical signals of Ag NCs recorded under such a situation are shown in Fig. 1A (curve b) and Fig. 1B (curves c and d).
Fig. 1 (A) UV-vis absorption spectra of the silver species synthesized with the probe (a), the probe–HBV gene duplex (b) and the HBV gene (c) as the scaffold, respectively. (B) Fluorescence excitation and emission spectra of silver species produced in different situations (a and b: probe; c and d: probe–HBV gene duplex; e and f: HBV gene).
4850 | Chem. Commun., 2014, 50, 4849--4852
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Note that the Ag NCs thus produced showed a slight shift in the characteristic absorption and emission maxima as compared with the 12-base template alone. This phenomenon is commonly observed in DNA-templated Ag NC synthesis and can be attributed to the change in the microenvironment of the DNA-Ag NCs.9 The generation of Ag NCs was also evidenced by the TEM image which clearly showed the existence of small particles with an average size of 1.88 0.25 nm (Fig. S3, ESI†). To exclude possible contribution from the HBV gene to the formation of Ag NCs, this single-strand DNA was also used as a template to confirm that no Ag NCs were produced (curve c in Fig. 1A and curves e and f in Fig. 1B), which is also evidence for the sequence-dependence of Ag NC growth. On the other hand, we also examined whether the growth of Ag NCs on the DNA scaffold would affect the stability of the already formed DNA duplex (recognition sequence-HBV gene) by polyacrylamide gel electrophoresis analysis (Fig. S4, ESI†). After the synthesis of Ag NCs on the formed duplex, the gel band was similar to the duplex without Ag NCs and no obvious band for ssDNA was observed, suggesting that the hybridized structure was not affected by the chemical reduction process. The above results indicated the effective locking of the nucleation sequence by the blocking segment as well as the successful release of the template upon the reaction of the target with the probe. Following the verification of the ability of this hairpin probe to control the in situ growth of Ag NCs, we further optimized the length of the blocking sequence. According to the principle of the probe design, the length of the blocking sequence can be a key factor to decide the signal-to-background ratio. A longer blocking sequence is able to hybridize with the nucleation sequence better and lock the probe more tightly, so that the portion of unlocked probe, i.e., the free template of Ag NCs will be reduced leading to a lowered background. On the other hand, however, the longer blocking sequence may hinder the unlocking of the probe, which will impair the release of the template and thus leads to a lowered signal in the target sensing process. Therefore, the length of the blocking sequence needs to be rationalized to balance between the two contrary effects. To this end, we further designed two probes (Table S1, probe 2 and 3, ESI†) containing the same recognition sequence and nucleation sequence as probe 1, while the length of the blocking sequence of probe 2 and 3 was 5 and 10 bases, respectively. Upon using the locked probes as templates to synthesize Ag NCs, the product of probe 2 with a 5-base blocking sequence exhibited strong absorption of Ag NCs, while the absorption of the other two probes was very weak (Fig. S5A, ESI†). The comparison of the fluorescence emission of these three systems also showed the same results (Fig. S5B, ESI†). It is concluded that considerable unlocked templates existed in the system of probe 2 due to the incomplete hybridization. It is also seen that the background of probe 1 (7-base) and probe 3 (10-base) did not differ too much, indicating that the hybridization between 7 pairs of nucleobases was basically adequate for the locking of the probe. The Ag NCs were synthesized by adding the same amount of target DNA to the three probes (Fig. 2). For probe 2, the Ag NC emission did not show much enhancement in the presence of
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Fig. 2 The fluorescence emission of Ag NCs produced on HBV gene probes (250 nM) with varying lengths (5, 7 and 10 bases) of blocking sequence. The black bars are the probes in their locking state as the template for Ag NC synthesis, while the red bars are the situations of probes in the presence of the same concentration (250 nM) of targets.
target DNA. Whilst the fluorescence of probe 3, as speculated, was much lower than the other two. This can be explained by the over strong hybridization between 10 base pairs which inhibited the release of the Ag NC template. In terms of the signal-to-background ratio which decides the sensitivity of assays, probe 1 with the 7-base blocking sequence afforded the best efficiency. As the cytosine-rich sequence may have an effect on the signal, we also designed two other target DNAs (Table S1, target 2 and 3 with 25 and 20 bases, respectively, ESI†) to optimize the target DNA sequence length. As shown in Fig. S6 (ESI†), decreasing the target sequence length resulted in a weakened signal and the optimal signal was obtained using the 30-base sequence as the target. We then investigated the performance of the probe in the detection of the HBV gene (target 1), using the probe with a 7-base blocking sequence. With the introduction of increasing amounts of HBV gene into the probe solution, the fluorescence emission intensity at 600 nm increased gradually (Fig. 3A), which, as expected, was a result of the formation of Ag NCs on the released nucleation sequence. The relative fluorescence intensity of Ag NCs ((F – F0)/F0, in which F and F0 represent the emission intensity of Ag NCs in the presence and in the absence of target molecules, respectively) was dependent on the concentration of HBV gene, and a linear calibration was obtained within 10–200 nM (Fig. 3B). The limit of detection, at a signal-to-noise ratio of 3.0,
Fig. 3 (A) The fluorescence emission of DNA–Ag NCs in the presence of varying concentrations (0, 10, 20, 40, 60, 80, 100, 120, 150, 200 nM) of HBV gene. (B) The linear relationship between the relative fluorescence intensity (F F0)/F0 and the concentration of HBV gene ranging from 10 to 200 nM.
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was estimated to be 3.0 nM. To evaluate the sequence specificity of this probe, we also investigated the response of the probe towards two other sequences, a single-base mismatched (SBM) sequence and a non-complementary (NC) sequence. From the results shown in Fig. S7 (ESI†), we could conclude that the Ag NC-based hairpin probe has the capability to distinguish target DNA from NC and SBM DNAs. We also detected the recovery of the HBV gene in spiked human serum samples. Three concentrations of the target (40, 80 and 120 nM) were spiked into 50-fold diluted human serum. The recovery values were in the range of 96–103% (Table S2, ESI†), indicating that the probe is capable of analysing real biological samples without interference. Thus far, we have demonstrated the feasibility of this novel hairpin DNA probe and validated its applicability in DNA sensing. In order to find out whether interactions other than the hybridization of oligonucleotides, such as targets that can alter the conformation of the probe, can also give out a sensing signal, another three-segment probe for the detection of the protein thrombin was constructed. The similar nucleation sequence and blocking sequence were used to form the hairpin probe (Table S1, ESI†), but a sequence of thrombin aptamer was embedded in the loop region (Fig. S8, ESI†). Considering the relatively large size of thrombin protein which might cause a steric hindrance preventing the formation of Ag NCs, we inserted a spacer containing nine T bases between the sequence of the Ag NC nucleation sequence and the thrombin aptamer. When the target protein was added to the probe and reacted with the aptamer, the conformation of the latter was changed leading to the unlocking of the probe. As a result, luminescent Ag NCs were generated via the reduction of silver ions directly in the solution. The Ag NCs produced in this case showed a characteristic absorption at 600 nm and a maximal fluorescence emission at 660 nm, respectively (Fig. S9, ESI†). The shifts in spectral maxima can be explained by the change in the microenvironment of the DNA–Ag NCs as mentioned above. Similarly, the introduction of thrombin resulted in the increase of the fluorescence emission at 660 nm (Fig. S10A, ESI†). The fluorescence intensity enhancement of the DNA–Ag NCs was dependent on the amount of thrombin introduced (Fig. S10B, ESI†). In a control experiment where some other species including proteins and amino acids were added, even with a concentration 30 times higher than that of thrombin, the emission at 660 nm showed no obvious change (Fig. S11, ESI†), which precluded possible influence of these species on the fluorescence emission and confirmed that the turn-on signal was a result of the formation of Ag NCs induced exclusively by the target. In summary, we have developed a novel hairpin DNA probe, which simply consists of three DNA sequences: a nucleation sequence as the template for Ag NC formation, a recognition sequence for specific targets and a short blocking sequence. The probe is based on target-induced release of the Ag NC template and subsequent in situ generation of Ag NCs. The designed probe is a simple single-stranded DNA without any modification or complex structure. By embedding different recognition sequences, the probes can be conveniently used for specific and versatile detection of diverse targets. This novel design may provide a simple and flexible strategy for nucleic acid-based molecular probe engineering.
Chem. Commun., 2014, 50, 4849--4852 | 4851
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This work was supported by the National Natural Science Foundation of China (No. 21075094, 21375098), the National Basic Research of China (973 program, No. 2011CB933600) and the Program for New Century Excellent Talents in University (NCET-11-0402).
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