Development of an Aptamer-Based Sensing Platform for Metal Ions

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Development of an Aptamer-based Sensing Platform for Metal Ions, Proteins and Small Molecules Through Terminal Deoxynucleotidyl Transferase-induced G-quadruplex Formation Ka-Ho Leung, Bingyong He, Chao Yang, Chung-Hang Leung, Hui-Min Wang, and Dik-Lung Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08314 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

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ACS Applied Materials & Interfaces

Development of an Aptamer-based Sensing Platform for Metal Ions, Proteins and Small Molecules Through Terminal Deoxynucleotidyl Transferase-induced G-quadruplex Formation Ka-Ho Leung,‡1 Bingyong He,‡1 Chao Yang,2 Chung-Hang Leung,*2 Hui-Min David Wang*3 and Dik-Lung Ma*1,4 1

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong E-mail:

[email protected] 2

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical

Sciences, University of Macau, Macao E-mail: [email protected] 3

Graduate Institute of Natural Products, Kaohsiung Medical, University, Kaohsiung 807, Taiwan

E-mail: [email protected] 4

Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist

University, Hong Kong, China ‡These authors contributed equally to this work.

ACS Paragon Plus Environment

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ABSTRACT We report a label-free, structure-independent luminescent sensing platform for metal ions, proteins and small molecules utilizing an Ir(III) complex, terminal deoxynucleotidyl transferase (TdT) and a structure-folding aptamer. A novel G-quadruplex-selective Ir(III) complex was identified to detect the nascent G-quadruplex motifs with an enhanced luminescence response. Unlike most label-free DNA-based assays reported in the literature, this sensing platform does not require a specific secondary structure of aptamer, thus greatly simplifying DNA design. The detection platform was demonstrated by the detection of K+ ions, thrombin and cocaine as representative examples of metal ions, proteins and small molecules. KEYWORDS: G-quadruplex; iridium(III) complex; label-free; TdT; universal detection


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INTRODUCTION Aptamers are DNA or RNA molecules that bind to specific targets with high affinity and selectivity. In theory, it is possible to discover an aptamer for any target via the SELEX (Systematic Evolution of Ligands by EXponential enrichment) strategy, leading to numerous reported applications of aptamers in the fields of chemotherapeutics, biosensing and bioimaging.1 Some aptamers undergo a change in secondary structure upon binding to their cognate targets, which allows them to function as recognition units for analytical assays. Aptamers have been combined with luminescent, colorimetric or electrochemical signal transducers to develop a wide range of DNA-based sensing platforms.2-3 Initial studies utilized DNA aptamers covalently linked fluorophore and quencher moieties for monitoring the structural change of DNA. However, the covalent labeling of DNA is expensive, and the modified aptamer may show reduced binding affinity and/or selectivity to the target analyte compared with native DNA. As a consequence, the label-free approaches that employ reversible luminescent probes to recognize specific DNA conformations have been explored.4 The G-quadruplex is a DNA secondary structure formed from guanine-rich sequences.5-6 The rich structural variety of the G-quadruplex motif7-8 has stimulated the development of numerous G-quadruplex-based assays for the detection of metal ions,9-10 DNA,11-14 small molecules,15-17 protein18-19 and enzyme activity.18,

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Meanwhile, terminal deoxynucleotidyl

transferase (TdT) is a DNA polymerase that catalyzes the polymerization of protruding, recessed or blunt-ended double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) molecules without requiring a template.24 As the result, the product sequence is nearly random and strongly dependent on the composition of substrate deoxyribonucleoside triphosphate (dNTP) pool. Interestingly, TdT could produce random G-quadruplex-forming sequences when a dNTP pool


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consisting of 60% dGTP and 40% dATP was used.25 The percentage of guanine required to form a 20-nucleotide G-quadruplex structure containing four G-tracts is thought to be about 50−70%. Inspired by these concepts, we report herein a DNA-based, label-free, structure-independent sensing platform for metal ions, proteins and small molecules utilizing a G-quadruplex-selective Ir(III) complex, TdT and a structure-folding aptamer. The mechanism of this assay is depicted in Scheme 1. In the absence of the target, the aptamer will be digested by ExoI in the 3' to 5' direction.26 Thus, TdT will not be able to produce the G-quadruplex sequences and the luminescence of the system will be weak due to the weak background emission of the Ir(III) complex. However, the addition of the target induces the folding of the aptamer into a secondary structure that resists digestion by ExoI. In the presence of a dNTP pool consisting of 60% dGTP and 40% dATP, TdT will then elongate the intact DNA oligonucleotide into a long, random, guanine-rich sequence. Upon the addition of K+ ions, the DNA will be induced into Gquadruplex structures that are subsequently recognized by the G-quadruplex-selective Ir(III) complex with a strong luminescence response, allowing the system to function as a switch-on luminescent probe for the target. We envisage that the use of the G-quadruplex-selective Ir(III) complex to recognize the TdT-induced G-quadruplex structures may allow our platform to provide similar sensitivity towards different targets in an aptamer structure-independent manner.


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EXPERIMENTAL SECTION G-quadruplex fluorescent intercalator displacement (G4-FID) assay. The G4-FID assay was employed to evaluate the G-quadruplex binding affinity of the test compound. Thiazole orange (TO) is a DNA probe that binds to G-quadruplex DNA (or dsDNA) with a strong luminescence response. The addition of an exogenous G-quadruplex binding compound will displace TO from the TO-G-quadruplex assemble, thus decrease the luminescence of the system. As the result, the magnitude of the decrease of luminescence reflects the degree of TO displacement, which is dependent on the G-quadruplex-binding ability of the test compound. The assay was performed as previously described.4 Fluorescence resonance energy transfer (FRET) melting assay. Fluorophore and quenchertagged-G-quadruplex (or dsDNA) sequences were employed in the FRET melting assay. The fluorophore and quencher are close to each other in the G-quadruplex structure, thus the luminescence of the beacon is suppressed. Upon heating, the G-quadruplex structure will be denatured and the distance between fluorophore and quencher will be increased, allowing the luminescence of the fluorophore to be recovered. An exogenous G-quadruplex-binding compound that stabilizes the G-quadruplex will increase the temperature required for the luminescence of the fluorophore to be recovered. As the result, the change of melting temperature of the G-quadruplex is correlated with the G-quadruplex-binding activity of the test compound. The FRET assay was performed as previously described.27 Synthesis. The following complexes were prepared according to (modified) literature methods.28-32 All complexes are characterized by 1H NMR,

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C NMR, high resolution mass

spectrometry (HRMS) and elemental analysis. Complex 1. 1H NMR (400 MHz, Acetone-d6) δ


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8.97 (d, J = 8.8 Hz, 2H), 8.91 (d, J = 8.8Hz, 2H), 8.15-8.05 (m, 8H), 7.90-7.86 (m, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.66-7.62 (m, 2H), 7.27-7.23 (m, 2H), 7.05-7.01 (m, 2H), 6.87 (dd, J1 = 1.6 Hz J2 = 8.0 Hz, 2H), 6.17 (d, J = 1.2 Hz, 2H), 2.43-2.29 (m, 4H), 1.02 (t, J = 7.6 Hz, 6H); 13C NMR (100 MHz, Acetone-d6) 168.5, 161.1, 151.3, 149.7, 149.0, 147.6, 142.1, 142.0, 139.3, 131.9, 131.4, 130.9, 129.9, 129.7, 129.2, 125.9, 123.6, 123.2, 122.9, 120.29, 29.8, 18.6; MALDI-TOFHRMS: Calcd. for C44H36IrN4 [M–PF6]+ : 813.2569 Found: 813.3104; Anal.: (C44H36IrN4PF6 +H2O) C, H, N: calcd. 54.15, 3.92, 5.74; found. 54.29, 3.8, 5.92. Complex 2. Reported.33 Complex 3. Reported.33 Detection of thrombin, cocaine and K+. To a 30 μL solution of Tris buffered solution (K+ detection: 50 mM Tris-HCl, 2 mM MgCl2, pH 8.3; thrombin detection: 100 mM Tris, 140 mM NaCl, 20 mM MgCl2, 20 mM KCl; cocaine detection: 25 mM Tris-HCl, 0.15 M NaCl, 2 mM MgCl2, pH 8.0) with the indicated concentration of target was added the appropriate aptamer (1 μM). Then, ExoI (K+ detection: 0.33 U/μL; thrombin detection: 0.33 U/μL; cocaine detection: 0.66 U/μL) was added and the samples were incubated at 37 °C for 30 min to allow DNA cleavage take place and the reaction was inactivated by heating at 80 °C for 20 min. 5 μL 10 × NEB TdT buffer, 250 μM CoCl2 2.4 mM dGTP, 1.6 mM dATP and 0.33 U/μL of TdT was added and the solution was made up with H2O to 50 μL. After 1 h in 37 °C, the reaction was stopped by the addition of 25 μL 100 mM EDTA-Na2. The samples were added to 425 μL TrisHCl buffer (20 mM Tris, 50 mM KCl, pH 7.2) and the resulting solutions were incubated for 30 min. Emission spectra were recorded in the 540−760 nm range using an excitation wavelength of 360 nm.


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RESULTS AND DISCUSSION In this study, three Ir(III) complexes (1–3, Figure 1) were examined for their luminescence response to different structure of DNA, including G-quadruplex, single-stranded DNA (ssDNA) and double-stranded DNA (ds17 and ds26) (Table S1). These three complexes share the same 2,2'-biquinoline (biq) N^N ligand, but contain different 2-phenylpyridine (ppy) derivatives as C^N ligands. Complexes 1 and 2 selectively displayed an enhanced luminescence response towards Pu27 G-quadruplex over ssDNA and dsDNA. Interestingly, complex 1, which differs from 2 only in the addition of an ethyl group at the 4ꞌ-position of the ppy C^N ligand, displayed the highest luminescent enhancement for G-quadruplex DNA out of the three complexes studied (Figure 1b). To investigate the role of the ethyl group, we performed G-quadruplex fluorescent intercalator displacement (G4-FID) and fluorescence resonance energy transfer (FRET) melting assays to compare the G-quadruplex binding affinity of complexes 1 and 2. The G4-FID assay showed that 2.0 µM of complex 1 was able to displace 50% of thiazole orange (TO) from TO-Gquadruplex assembly (G4DC50 = 2.0 µM, half-maximal concentration of compound required to displace 50% TO from DNA) with slightly higher efficacy than complex 2 (G4DC5 = 2.3 μM, Figure 1c). Additionally, FRET-melting assays revealed that the melting temperature (Tm) of the F21T G-quadruplex was increased by about 7.8 °C and 19.4 °C in the presence of 3 and 5 μM of complex 1, respectively (Figure 1d). By comparison, the Tm was increased by smaller values of about 7.6 °C and 13.9 °C upon the addition of 3 μM and 5 μM of complex 2, respectively. These results outline that the presence of ethyl group in complex 1 slightly increases the G-quadruplexbinding affinity of complex 1. Interestingly, complex 3, which differs from complex 2 only in the presence of an additional aldehyde group, displayed no selectivity towards G-quadruplex over either ssDNA or dsDNA, which we attribute to the weak binding of complex 3 towards G-


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quadruplex DNA as confirmed by G4-FID (G4DC5 > 6 μM) and FRET melting assays (Tm = 2 °C at 5 μM). These results indicate that the aldehyde group significantly detracts from Gquadruplex-binding affinity, thus leading to a poor luminescence selectivity for G-quadruplex DNA over ssDNA or dsDNA. This result is consistent with our previous experience, in which the binding affinity of metal complexes may be fine-tuned by the small modifications to the auxiliary ligands.34 We next examined the luminescence response of complex 1 towards a variety of G-quadruplex structures. Complex 1 showed a relative high luminescence towards c-myc and c-kit Gquadruplex sequences (Pu27, Pu22, c-myc, c-kit1, ckit87up), but a relatively lower enhancement (ca. 2.7 fold) for the human telomeric G-quadruplex sequence (Figure S1b). On the other hand, no significant enhancement was observed for the thrombin binding aptamer (TBA), dsDNA and ssDNA. The strong luminescence enhancement of complex 1 is presumed to result from the shielding of the iridium center from water-mediated quenching upon G-quadruplex binding, thereby preventing radiationless decay of the excited state and producing 3MLCT emission. The G-quadruplex-binding selectivity of complex 1 was further evaluated using G4-FID and FRET melting assays. The results revealed that complex 1 could displace 50% of TO from Pu27, Pu22 and ckit87up at a concentration of 2 µM, whereas >5 μM of 1 was required for duplex DNA (Figure S1c). Additionally, complex 1 increased the Tm of the F21T G-quadruplex by about 7.8 °C (Figure S1d), but only increased the Tm of F10T dsDNA by 1.5 °C under the same conditions (Figure S1e). Finally, the stabilization effect of complex 1 towards the F21T Gquadruplex was not significantly influenced by the addition of 50-fold higher concentration of unmodified dsDNA (ds26) or ssDNA (Figure S1f). Taken together, these results demonstrate the ability of complex 1 to selectively bind G-quadruplex DNA over dsDNA or ssDNA.


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Complex 1 shows a weak luminescence for the TBA G-quadruplex, we reasoned that it may bind outside the G-tetrad as previously reported that TBA only effectively bind to the planar aromatic ligands.4 To further explore the binding location of complex 1, we investigated its interaction with G-quadruplexes containing different loop sizes in the central loop. The luminescence of complex 1 increased with central loop size from 2 to 15 nt, suggesting that the loop region may play a significant role in the binding activity. The unusually high luminescence enhancement with the central loop containing 1 nt may be due to its ability to form an intermolecular G-quadruplex structure. Based on the effectiveness of 1 as a G-quadruplex probe, we sought to employ 1 as a signal transducer for the aptamer-based detection platform. To validate the feasibility of the proposed method, we chose K+ ions as a model for metal ion detection. K+ ions play an important role in biological systems, such as in the maintenance of extracellular osmolarity and the production of electrical signals in nerve systems.35 The G-quadruplex Pu22 was used as the recognition unit for K+ detection, and the assay was performed as outlined in Scheme 1. We found that the luminescence of complex 1 was enhanced with increasing concentrations of K+ ions. On the other hand, no enhancement was observed in the absence of DNA, indicating that K+ ions did not directly interact with complex 1 (Figure S3). Moreover, the luminescence enhancement of the system in response to K+ ions was significantly smaller in the absence of TdT (Figure S4). We believe that the residual luminescence enhancement of the system when TdT is absent is due to the K+-induced G-quadruplex formation of Pu22. We also investigated the signal output of a system using a mutant Pu22 sequence that is unable to bind to K+ ions. No luminescence enhancement was observed upon the addition of K+ ions to the modified system (Figure S5), suggesting that K+-Pu22 interaction is involved in the mechanism of the assay. Additionally, no


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significant enhancement was recorded when a 100% dTTP pool was used (Figure S6), suggesting that the formation of the G-quadruplex motif by TdT is required for luminescent enhancement. To obtain the highest sensitivity of the assay, we investigated the effect of several parameters on the luminescence response of the system. It was observed that increasing the concentration of TdT beyond 0.4 U/μL offered no significant enhancement in luminescence (Figure S7). As K+ ions are involved in the stabilization of the G-quadruplex structures produced by TdT, the effect of KCl concentration on the luminescence response of the system was also examined. The results showed that the maximal luminescence response of the system was obtained at 50 mM of KCl (Figure S8). Finally, as ExoI is added to digest the unbound aptamer, we sought to determine the concentration of ExoI needed for maximal DNA cleavage. The nonspecific DNA dye SYBR Green I (SG) was employed to quantify the amount of undigested DNA. The fluorescence intensity of SG decreased with increasing concentrations of ExoI, with a maximal decrease reached at 0.66 U/μL of ExoI (Figure S9). Thus, 0.66 U/μL of ExoI was considered sufficient to cleave the maximal amount of unbound Pu22 aptamer. We then performed an emission titration experiment with increasing concentrations of K+ using the optimized conditions. Encouragingly, the luminescence intensity of complex 1 was enhanced as the concentration of K+ was increased (Figure 2a–b). The system exhibited a linear range of detection for K+ from 1 to 100 μM, with a ca. 6-fold maximal luminescence enhancement (Figure 2c). The detection limit was determined to be 0.4 μM by the 3σ method. The selectivity of our approach for K+ ions was evaluated by testing the response of the system to nine other metal ions (Na+, Ni2+, Ca2+, Zn2+, Mg2+, Pb2+, Fe3+, Cu2+ and Li+). The results showed that only K+ could significantly enhance the luminescence of the complex 1/G-


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quadruplex system (Figure 2d), while no significant change in emission intensity was observed upon the addition of the other metal ions. Notably, Na+ induced only about 15% of the relative enhancement compared to K+ which agrees with a previous study by Lou and co-workers.36 We attribute this result to the weaker ability of Na+ to stabilize the Pu22 aptamer compared to K+. To validate the broad versatility of our sensing platform, we evaluated the application of the system to detect thrombin as an example of protein detection. The thrombin aptamer undergoes a structural change from ssDNA to G-quadruplex DNA upon binding to thrombin. As before, we initially determined the optimal concentration of ExoI required to fully digest the unbound aptamer (Figure S10). Next, the luminescence intensity of complex 1 was enhanced as the concentration of thrombin was increased (Figure 3a−b). The system exhibited a linear range of detection for thrombin from 0.5 to 15 nM with detection limit of 0.1 nM as determined by 3σ method (Figure 3c). Furthermore, the luminescence of complex 1 was unaffected by the sole addition of thrombin, revealing that the luminescence enhancement of the system originated from specific interaction of complex 1 with G-quadruplex DNA (Figure S11). Additionally, the sensitivity of the platform was significantly decreased in the absence of TdT (Figure S11). Finally, no luminescence enhancement was observed investigating the response of a modified system involving a mutant TBA that cannot bind to thrombin (Figure S12), suggesting that thrombin-TBA interaction is important for the mechanism of the assay. The selectivity of our approach for thrombin was evaluated by investigating the response of the system to four other proteins (IgG, IgM, IgA and human neutrophil elastase). A significant luminescent enhancement was only observed with thrombin, but not the four other proteins (Figure 3d). After successfully demonstrating the monitoring of metal ions and proteins, our detection platform was next applied for the sensing of cocaine, a small molecule. Unlike the K+ and


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thrombin aptamers, the cocaine aptamer folds into a three-way junction-like DNA structure upon ligand binding to cocaine. We hypothesized that this DNA structure could similarly resist cleavage by ExoI, thereby allowing the TdT-mediated production of random G-quadruplex sequence. Interestingly, we observed that twice the concentration of ExoI was required to achieve full cleavage of cocaine aptamer (Figure S13), which may due to partial pre-folding of the aptamer. In the cocaine detection assay, the luminescence intensity of complex 1 was enhanced as the concentration of cocaine was increased (Figure 4a‒b). The system exhibited a linear range of detection for cocaine from 0.5 to 25 μM (Figure 4c) and detection limit is 0.3 μM which determined by 3σ method. In a control experiment, the luminescence of complex 1 was not enhanced upon the sole addition of cocaine (Figure S14). Furthermore, no luminescence enhancement was observed upon addition of cocaine using mutant cocaine aptamer sequence that is unable to bind to cocaine (Figure S15), suggesting that the cocaine-aptamer assembly is important for the mechanism of the assay. Finally, no significant enhancement was observed when cocaine was added to a detection system containing a 100% dTTP pool (Figure S16). The selectivity of our approach for cocaine was evaluated by investigating the response of the system to four other small molecules (ATP, adenosine, warfarin and suramin). The results showed that only cocaine significantly increases the luminescence signal of the system (Figure 4d). Taken together, the results with K+, thrombin and cocaine indicate the assay is independent of the secondary structure of the aptamer employed to detect the target analyte. Circular dichroism (CD) spectroscopy was used to study the structure-switching behavior of DNA in this detection platform. We first incubated ssDNA with TdT in the presence of a dNTP pool consisting of 60% dGTP and 40% dATP. The CD spectrum of the product exhibited an intense positive peak at around 267 nm and a strong negative peak at 245 nm (Figure 5),


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consistent with the randomly-arrayed G-quadruplex previously reported.36 Moreover, all aptamers displayed an increase in signal change at 270 nm upon addition of K+, thrombin or cocaine, respectively, indicating the formation of randomly G-quadruplexes. This suggests that the luminescence enhancement in the presence of the target can be attributed to the formation of the G-quadruplex motif. We also investigated the application of our sensing ensemble under a model of physiological conditions. An oral fluid sample was collected from a volunteer and diluted by 50-fold using Tris buffer (20 mM Tris, pH 7). The system experienced a gradual increase in luminescence intensity as the amount of spiked cocaine was increased (Figure S17). Next, we investigated the performance of our sensing platform for thrombin in the presence of cellular debris. If Gquadruplex or other DNA secondary structures were present in significant quantities in cell extracts, this could generate a high background signal that could reduce the sensitivity of the assay. We therefore compared the luminescence intensity of our sensing platform in aqueous buffer versus 0.5% v/v cell extract in the absence of the target. No significant increase of the luminescence intensity was observed in the diluted cell extract compared to the aqueous buffer system, suggesting that the cell extract did not contain significant quantities of G-quadruplex or other DNA secondary structures that could lead to false positive results (Figure S18). The subsequent addition of thrombin to the reaction system containing 0.5% v/v cell extract induced a gradual increase in luminescence intensity (Figure S19), in a similar fashion compared to the operation of the detection platform in aqueous buffer. This result indicates that our sensing platform was not significantly perturbed by the addition of diluted cell extract, and demonstrates that this assay could potentially be further developed for analyte detection in biological samples. CONCLUSION


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In conclusion, three luminescent Ir(III) complexes containing different C^N ligands were investigated for their ability to act as G-quadruplex-selective probes. The novel Ir(III) complex 1 showed the best selectivity for G-quadruplex DNA compared to the other complexes tested. Complex 1 was then used to develop a DNA-based, label-free, structure-independent sensing platform. Unlike most label-free DNA-based universal detection assays reported in the literature, this sensing platform does not require a specific secondary structure of aptamer or any specific DNA sequence design, thus greatly simplifying DNA design. The label-free approach also prevents the use of expensive modified DNA that could detect targets in low cost. For easier access, recently reported universal detection platforms are shown in Table S2 for comparison. The structure-independent sensing platform was demonstrated by the detection of K+ ions, thrombin and cocaine as representative examples of metal ions, proteins and small molecules, respectively, and could be potentially utilized in biological sample detection. Moreover, this study represents, to our knowledge, the first example of a Ir(III) complex being applied for the development of a detection platform for metal ions, proteins and small molecules with signal amplification. As similar G-quadruplexes are produced by TdT for each target, we envisage that the assay can provide similar sensitivity for different kinds of target in an aptamer structuredependent fashion.

Supporting information Experimental section; DNA sequences used in this project; optimization data; real sample detection; These experimental data in this study is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by Hong Kong Baptist University (FRG2/14-15/004), the Health and Medical Research Fund (HMRF/13121482 and HMRF/14130522), the Research Grants Council (HKBU/201811, HKBU/204612 and HKBU/201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12), State Key Laboratory of Environmental and Biological Analysis Research Grant (SKLP-14-15-P001), National Natural Science Foundation of China (21575121), Guangdong Province Natural Science Foundation (2015A030313816), Hong Kong Baptist University Century Club Sponsorship Scheme 2015, Interdisciplinary Research Matching Scheme (RC-IRMS/14-15/06), the State Key Laboratory of Synthetic Chemistry, the Science and Technology Development Fund, Macao SAR (103/2012/A3 and 098/2014/A2), the University of Macau (MYRG091(Y3L2)-ICMS12-LCH, MYRG2015-00137-ICMS-QRCM and MRG023/LCH/2013/ICMS)

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TOC FIGURE


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Scheme 1. Schematic diagram of the label-free randomly arrayed G-quadruplex-based detection platform utilizing a G-quadruplex-selective Ir(III) complex, TdT and a structure-folding aptamer. 254x190mm (300 x 300 DPI)


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Figure 1. (a) Chemical structures of the Ir(III) complexes 1–3 investigated in this study. (b) Luminescence enhancement of complexes 1–3 (1 µM) in the presence of 3 µM of ssDNA, ds17, ds26 or Pu27 G-quadruple. (c) G4-FID titration curves of complexes 1–3 against the Pu27 G-quadruplex. (d) Melting profiles of F21T Gquadruplex DNA (0.2 µM) in the absence and presence of 3 or 5 µM of complexes 1–3. 593x306mm (300 x 300 DPI)



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Figure 2. (a) Luminescence spectra of the system in response to increasing concentrations of K+. (b) The relationship between luminescence intensity at λ = 637 nm and K+ concentration. (c) Linear plot of the change in luminescence intensity at λ = 637 nm vs. K+ concentration. (d) Relative luminescence intensity of the system in the presence of 100 µM K+ or other metal ions. 179x140mm (300 x 300 DPI)


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Figure 3. (a) Luminescence spectra of the system in response to increasing concentrations of thrombin. (b) The relationship between luminescence intensity at λ = 637 nm and thrombin concentration. (c) Linear plot of the change in luminescence intensity at λ = 637 nm vs. thrombin concentration. (d) Relative luminescence intensity of the system in the presence of 3 nM thrombin or other proteins. 179x137mm (300 x 300 DPI)



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Figure 4. (a) Luminescence spectra of the system in response to increasing concentrations of cocaine. (b) The relationship between luminescence intensity at λ = 637 nm and cocaine concentration. (c) Linear plot of the change in luminescence intensity at λ = 637 nm vs. cocaine concentration. (d) Relative luminescence intensity of the system in the presence of 25 µM cocaine of other small molecules. 179x139mm (300 x 300 DPI)


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Figure 5. Circular dichroism (CD) spectrum of (a) 1 µM of ssDNA upon incubation with TdT in the presence of 6:4 dGTP:dATP. Circular dichroism (CD) spectra of the sensing platform with 1 µM of (b) Pu22, (c) cocaine aptamer and (d) TBA in the absence (blue) or presence (red) of K+, cocaine and thrombin, respectively. 169x116mm (300 x 300 DPI)


Development of an Aptamer-Based Sensing Platform for Metal Ions

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