A General Chemiluminescence Strategy for Measuring Aptamer

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A General Chemiluminescence Strategy for Measuring Aptamer− Target Binding and Target Concentration Shiyuan Li, Duyu Chen, Qingtong Zhou, Wei Wang, Lingfeng Gao, Jie Jiang, Haojun Liang, Yangzhong Liu, Gaolin Liang, and Hua Cui* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China S Supporting Information *

ABSTRACT: Although much effort has been made for studies on aptamer−target interactions due to promising applications of aptamers in biomedical and analytical fields, measurement of the aptamer−target binding constant and binding site still remains challenging. Herein, we report a sensitive label-free chemiluminescence (CL) strategy to determine the target concentration and, more importantly, to measure the target− aptamer binding constant and binding site. This approach is suitable for multiple types of targets, including small molecules, peptides, and proteins that can enhance the CL initiated by N(aminobutyl)-N-ethylisoluminol functionalized gold colloids, making the present method a general platform to investigate aptamer−target interactions. This approach can achieve extremely high sensitivity with nanogram samples for measuring the target−aptamer binding constant. And the measurement could be rapidly performed using a simple and low-cost CL system. It provides an effective tool for studying the binding of biologically important molecules to nucleic acids and the selection of aptamers. Besides, we have also discovered that the 14-mer aptamer fragment itself split from the ATP-binding aptamer could selectively capture ATP. The binding constant, site, and conformation between ATP and the 14-mer aptamer fragment were obtained using such a novel CL strategy and molecular dynamic simulation.

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the investigation of aptamer−target interactions. They can be divided into two categories.8 One is separation based techniques such as dialysis,9,10 ultrafiltration,11,12 gel and capillary electrophoresis,13,14 and high performance liquid chromatography (HPLC).15,16 The other involves mixture based techniques such as nuclear magnetic resonance (NMR),17,18 isothermal titration calorimetry (ITC),19 atomic force microscopy (AFM),20 quartz crystal microbalance (QCM),21 surface plasmon resonance (SPR),22 polymerase chain reaction (PCR) with radio detection,23 flow cytometry, and fluorescence assay (e.g., Förster resonance energy transfer based fluorescence method, fluorescence anisotropy/polarization).24−27 Although such approaches have their own merits, they also suffer from some defects.8,28 For example, they require either high sample consumption or labeling of target/ aptamer or separation after the recognition event or use of bipartite aptamers. Some approaches are difficult to use for measuring small molecule−aptamer binding such as QCM21 and SPR.22 Moreover, most approaches involve cumbersome

he binding of biologically important molecules, such as small molecules, peptides, and proteins, to nucleic acids is critically important in both fundamental research and applications in life sciences. Of particular interest are aptamers, i.e., short single-stranded nucleic acids (DNA or RNA). Aptamers can bind with high affinity and specificity to a wide range of target molecules, such as ions, small organic molecules, proteins, and cells.1−3 The high affinity of aptamers for their targets is afforded by their capability to fold upon binding their cognate target molecules. In particular, they can either incorporate small molecules into their nucleic acid structure or integrate into the structure of larger molecules, such as proteins. Compared with antibodies, aptamers exhibit a number of advantages. Aptamers can be routinely produced by chemical synthesis, avoiding the use of animals for antibody production. They are generally more chemically stable, and their binding properties are easier to manipulate. These features hold great promise in therapeutic, diagnostic, and analytical applications.4−7 In order to use an aptamer in research and clinical applications, a thorough understanding of aptamer−target binding is necessary. Studies on aptamer−target binding, especially those involving binding constants and binding sites, are very challenging. Various methods have been proposed for © 2014 American Chemical Society

Received: March 24, 2014 Accepted: May 14, 2014 Published: May 14, 2014 5559

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kept at 4 °C. All other reagents were of analytical grade. Ultrapure water was prepared by a Milli-Q system (Millipore, France) and used throughout. UV Absorption Spectrum Measurements. The 14-mer fragment (5′-ACCTGGGGGAGTAT-3′) split from the ATP binding aptamer was synthesized and HPLC-purified by Sangon Inc. (Shanghai, China). ATP and the 14-mer fragment were solvated in an aqueous solution containing 50 mM NaCl, 20 mM phosphate (pH 7.4). The final concentration of ATP was 10 μM, and that of the 14-mer fragment was 1 μM. UV absorption spectra of PBS buffer solution, ATP, the 14-mer fragment, and the mixtures of ATP and the 14-mer fragment were measured on a model UV-8453 UV−visible spectrometer (Agilent, USA). NMR Measurements. The 14-mer fragment used in the NMR was synthesized and PAGE-purified by Sangon Inc. (Shanghai, China). The concentration of the 14-mer fragment was 5 mM in a solvent of 90% H2O/10% D2O containing 50 mM NaCl, 20 mM phosphate (pH 7.4). ATP was added gradually to the 14-mer fragment solution from 1.0, 2.0, 5.0 to 10.0 equiv of the 14-mer fragment. The exchangeable proton NMR spectra of the 14-mer fragment (as control) and the complex of ATP and the 14-mer fragment were collected on a Bruker AV-600 (600 MHz) spectrometer. Preparation of ABEI−Au Colloid. The ABEI-functionalized gold colloid used in this work was obtained by a seed growth method as described previously.34 Briefly, a 9 mL portion of HAuCl4 stock solution was mixed with 45 mL of ultrapure water. While stirring vigorously, 9 mL of 4.0 mM ABEI solution were added rapidly, and the solution was maintained at room temperature for 2 h. Then another 6 mL portion of HAuCl4 stock solution was added. The reaction continued for another 2 h, and the ABEI-Au colloid was obtained. The prepared ABEI-Au colloids were stored at 4 °C for future use. CL Measurements. 100 μL aliquots of biotinylated 14-mer aptamer fragment 5′-biotin-ACCTGGGGGAGTAT-3′ (biotinF) or other biotinylated aptamer in 0.02 M PBS (pH 7.4) solution were injected into each SA-coated microwell, incubated at 37 °C for 2 h, and rinsed with 0.01 M Tris-HCl (pH 7.0, containing 0.1 M NaCl). A PST-60 HL plus Thermo Shaker (Biosan, Latvia) was used to control the temperature of the reactions. Then 100 μL aliquots of ATP or other target solution in 0.02 M PBS (pH 7.4) were added to each well, incubated at 37 °C for 2 h, and rinsed with 0.01 M Tris-HCl (pH 7.0, containing 0.1 M NaCl) to remove unbound ATP or other target molecules. Finally, 100 μL aliquots of the ABEI− Au colloid were injected into each well and incubated at 37 °C for 30 min before testing. CL measurements were performed with a microplate luminometer (Centro LB 960, Berthold, Germany) equipped with two injectors. When 100 μL of 0.15 M H2O2 in 0.1 M NaOH (pH 13.0) were injected into a well immobilized with a target−binding aptamer complex, the CL signal was recorded. The measurement time was optimized as 10 s with a time interval of 0.01 s. MD Simulation Parameters. The Gromacs 4.5.3 software package35 was used to perform an MD simulation with the AMBER03 force field.36 The initial coordinates of the 14-mer DNA aptamer fragment were extracted from the AMP−DNA aptamer complex (PDB code 1AW437), considering crystallographic or NMR data are not available. The system was solvated with 11769 TIP3P water molecules and neutralized

procedures, sophisticated instrumentation, and a long measuring time. Although a large number of aptamers have been found for proteins, relatively few aptamers have been available for small molecules. The aptamers for adenosine triphosphate (ATP) and its analogues such as adenosine monophosphate (AMP), cyclic-AMP, adenosine diphosphate (ADP), and adenosine are the most studied aptamers targeting small molecules.29 The aptamer for ATP was originally selected as a single strand DNA. Later on, the aptamer for ATP was split into two pieces, which has an equilibrium between its two dissociated parts and a folded, associated complex. When the target molecules bind to the complex selectively, the equilibrium will greatly be driven toward the complex form. By virtue of labeling or nonlabeling protocols, the detection of small molecules and small molecule−aptamer binding becomes possible.20,30−33 In these cases above, it was previously believed that the two split DNA strands were concurrently required to bind small molecules. In the present work, the interaction between ATP and one DNA fragment (5′-ACCTGGGGGAGTAT-3′) split from an ATP-binding aptamer (5′-ACCTGGGGGAGTATTGCGGAGGAAGGT- 3′) was studied using UV absorption and NMR spectra. It was found that the 14-mer aptamer fragment alone was sufficient to selectively capture ATP. By using a N-(aminobutyl)-N-(ethylisoluminol) functionalized gold colloid (ABEI−Au colloid) as a signal reporter and a microplate as an immobilizing substrate, a sensitive label-free chemiluminescence (CL) strategy has been proposed to measure the ATP−aptamer binding constant and binding site as well as determine ATP concentration. By virtue of such a novel CL approach, the dissociation constant of the ATP binding14-mer aptamer complex, the possible binding site of the 14-mer aptamer fragment responsible for ATP, and ATP concentration were determined. Moreover, in order to investigate the binding mechanism and conformation of ATP and the 14-mer aptamer fragment, molecular dynamic (MD) simulation was performed. The binding conformation and the binding pocket of the ATP binding 14-mer aptamer complex from simulation results were strongly supported by the results measured by the CL approach. Finally, the generalization of the proposed CL strategy for multiple types of targets and corresponding aptamers was also explored by taking nineamino acid cyclic hormone peptide arginine vasopressin (AVP) and protein thrombin (TB) and their corresponding aptamers as models, revealing that it is a general strategy for measuring target−aptamer binding and target concentration from small molecules to biomacromolecules.

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EXPERIMENTAL SECTION Chemicals and Solutions. SA, bovine serum albumin (BSA), ATP, uridine triphosphate (UTP), guanosine triphosphate (GTP), thymine (T), and cytosine (C) were all obtained from Solarbio (Beijing, China). Arginine vasopressin (AVP) and thrombin (TB) were obtained from Sangon Inc. (Shanghai, China). White 96-well plates were obtained from Pierce (USA). The aptamers used in this work were synthesized by Sangon Inc. (Shanghai, China), and the sequences are shown in Table S1. A HAuCl4 stock solution (2‰ HAuCl4, w/w) was prepared by dissolving 1.0 g of HAuCl4·4H2O (Shanghai Reagent, China) in 412 mL of purified water and stored at 4 °C. N(Aminobutyl)-N-(ethylisoluminol) (ABEI) was purchased from Sigma-Aldrich (USA). A 4.0 mM stock solution of ABEI was prepared by dissolving ABEI in 0.1 M NaOH solution and was 5560

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with Na+ ions in a box, which extended to at least 10 Å between the complex and the boundary of the box. All bonds were constrained using the LINCS algorithm. Long-range electrostatic interactions were computed using the Particle Mesh Ewald method;38 van der Waals and short-range electrostatic interactions were calculated with a cutoff of 10 Å. The entire system was first relaxed using the steepest descent energy minimization, followed by slow heating of the system to 300 K with restraints, and then the restraints were reduced gradually over 150 ps. Finally, the system was run for 70 ns without restraints with a time step of 2 fs in the NPT ensemble at 300 K and 1 bar using a Berendsen thermostat and barostat.

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RESULTS AND DISCUSSION Interaction between ATP and the 14-mer Aptamer Fragment. The interaction between ATP and a single 14-mer DNA fragment (5′-ACCTGGGGGAGTAT-3′) split from an ATP aptamer (5′-ACCTGGGGGAGTATTGCGGAGGAAGGT-3′) was studied by UV absorption spectra. As shown in Figure 1, it could be seen that the 14-mer fragment and ATP

Figure 2. 1D 1H NMR spectra of imino protons (9.0−12.5 ppm) of the 14-mer aptamer fragment (a) titrated by ATP (b−e). The ratio of ATP over 14-mer aptamer fragment is labeled in each spectrum.

ATP. This was different from previous reports that two split DNA fragments from the ATP aptamer were concurrently required to bind ATP. CL Measuring Principle. By taking the split fragment as the recognition element, ATP as a model target, N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold colloid (ABEI− Au colloid) as a signal reporter, and a microplate as an immobilizing substrate, a simple CL strategy was developed for measuring the 14-mer DNA fragment-ATP binding and ATP concentration as shown in Figure 3a. The 5′ termini of the split aptamers were biotinylated to form biotin-F (5′-biotinACCTGGGGGAGTAT-3′), followed by attachment to a 96well microplate via biotin−streptavidin (SA) interaction to serve as capture probes. Next, these immobilized capture probes interacted with ATP and, finally, with ABEI−Au colloids. When H2O2 was introduced into the system, CL was produced, which resulted from the reaction between ABEI in ABEI−Au colloids and H2O2.39 The CL reaction conditions were optimized as shown in Supporting Information section S1. The optimal conditions were as follows: 0.1 μM biotin-F, pH 13.0, 0.15 M H2O2, and 30 min incubation time. The relationship between CL intensity and the logarithm of ATP concentration is shown in Figure 3b. It was found that CL intensity increased linearly with the logarithm of ATP concentration at lower ATP concentrations ranging from 0.1 nM to 0.1 μM. The linear response of the logarithm of ATP concentration could be fitted by the equation ICL = 7.72856 × 105 + 5.1072 × 104 × log C, where ICL is the total CL intensity measured in 10 s, and C is the concentration of target ATP, with a correlation coefficient of R = 0.9954, as shown in Figure 3b (upper inset). This CL strategy could therefore be used to determine ATP. The detection limit at a signal-to-noise ratio of 3 (S/N = 3) for ATP was 28 pM. The reproducibility was evaluated by detecting ATP a total of 7 times, and the relative standard deviation of ATP concentration was 2.3% at 10 nM (n = 7), indicating good reproducibility. When the ATP concentration was over 0.1 μM, the CL intensity deviated from the linear relationship. The estimate of the binding fraction of the 14-mer aptamer fragment for ATP indicated that the binding fraction below 0.03 μM ATP was less than 3.2% and could thus be ignored (Supporting Information section S2). According to the principle underlying the measurement of the binding constant,20,24−27 the curve from 0.1 to 1 μM was applied. Equilibrium analysis was carried out at different ATP concentrations, and a sigmoid fitted curve was obtained as shown in Figure 3b (lower inset), which led to a

Figure 1. UV absorption spectra. (a) PBS; (b) ATP + PBS; (c) 14-mer fragment + PBS; (d) sum of (c) + (b) − (a); (e) 14-mer fragment + ATP + PBS. Final concentration of each reagent: 1 μM 14-mer fragment, 10 μM ATP, 0.02 M PBS.

had a notable absorption peak at 256 nm. When the 14-mer fragment was mixed with ATP, the intensity of the mixed system’s absorption spectra was not equal to the sum of the two individuals, indicating that the 14-mer fragment interacted with ATP. The interaction between ATP and the 14-mer fragment was also studied through NMR. The exchangeable proton NMR spectrum is shown in Figure 2. We observed a decrease in signal intensity and upfield chemical shift in the range from 12.5 to 9.0 ppm with the increase of ATP concentration from 1.0, 2.0, 5.0, to 10.0 equiv of the 14-mer fragment. The results demonstrated the possible conformational changes and rupture of hydrogen bonds of the 14-mer fragment after the addition of ATP, indicating the binding of ATP to the 14-mer fragment and forming of the ATP−DNA complex. Besides, adding ATP beyond 2.0equiv of the 14-mer fragment still rendered the formation of more ATP−DNA complexes, indicating that the association constant of ATP and the 14-mer fragment is moderate, because a weak binding would not cause an observable chemical shift change and a strong binding would result in the saturation of binding below 2.0 equiv of the 14mer fragment. The results demonstrated that the 14-mer fragment of the split DNA aptamer alone is able to capture 5561

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Figure 4. Effect of targets ATP, AVP, and TB on the CL reaction between the ABEI−Au colloid and H2O2. Reaction conditions: 100 μL, pH 13, 0.15 M H2O2.

aptamer increased with ATP concentration, leading to a linear increase in CL intensity. When the ATP concentration exceeded a threshold, the amount of ATP captured by the aptamer reached saturation, generating a constant CL intensity. The selectivity of the proposed CL strategy was also studied by control experiments using ATP analogues, such as 0.1 μM uridine triphosphate (UTP), guanosine triphosphate (GTP), thymine (T), and cytosine (C), instead of ATP as targets. As shown in Figure 5, ATP exhibited the strongest CL response,

Figure 3. CL measurement of aptamer-target binding and target concentration. (a) Schematic illustration of the proposed CL strategy. (b) Relationship between CL intensity and logarithm of ATP concentration. Upper inset: working curve for determination of ATP. Lower inset: measurement of dissociation constant of ATPbinding split aptamer.

Figure 5. Selectivity of the CL strategy for ATP. Concentration for ATP, UTP, GTP, T, and C: 0.1 μM. Reaction conditions: 100 μL, pH 13, 0.15 M H2O2.

while its analogues only showed weak CL signals. These results revealed that few ATP analogues could be captured by the 14mer aptamer fragment. Thus, the proposed strategy could distinguish ATP from its analogues. The good selectivity of this approach arose from the high selectivity of the 14-mer aptamer fragment. Moreover, the selectivity of the 14-mer aptamer fragment toward ATP is similar to that of the two split DNA fragments obtained by Xu’s electrochemiluminescence method.40 Therefore, the 14-mer aptamer fragment alone could selectively capture ATP. Binding Site Study. Thus far, it has been demonstrated that the proposed CL strategy could be used for measuring ATP−aptamer binding, implying the additional possibility of studying the binding site by sequentially mutating the aptamer bases. From the binding constants of ATP and mutated aptamers, the possible binding area of the aptamer responsible for the specific interactions can be determined. To investigate this important parameter, 0.1 μM DNA sequence G9 (5′biotin-ACCTGGGGTAGTAT-3′), G8 (5′-biotin-ACCTGGGTGAGTAT-3′), G7 (5′-biotin-ACCTGGTGGAGTAT3′), G6 (5′-biotin-ACCTGTGGGAGTAT-3′), A1 (5′-biotin-

dissociation constant of 0.65 μM. This value is consistent with that roughly estimated by NMR (Figure 2). Moreover, the dissociation constant of the ATP binding 14-mer aptamer fragment (0.65 μM) is comparable with those of the ATP binding 27-mer aptamer (6 ± 3 μM)17 or two split DNA fragments (3.7 μM),20 indicating that the binding ability of ATP with the 14-mer aptamer fragment is similar to that with the 27-mer aptamer or two split DNA fragments. It was observed that the CL intensity increased linearly with the logarithm of ATP concentration at lower concentrations, but maintained a nearly constant intensity beyond a certain ATP concentration. In order to validate the change of the CL intensity with the logarithm of ATP concentration, the effect of ATP on the CL reaction between the ABEI−Au colloid and H2O2 was studied by mixing the ATP solution with the ABEI− Au colloid and then injecting H2O2 into the mixture (Figure 4). With the increase of ATP concentration from 0 to 0.1 mM, the CL intensity increased significantly, indicating that the presence of ATP could enhance ABEI−Au colloid-initiated CL. At lower ATP concentration, the amount of ATP captured by the 5562

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Figure 6. CL measurement of dissociation constant of mutated DNA sequences. (a) G8, (b) G7, (c) G6, (d) A1, (e) C3, (f) A10, (g) G9, and (h) S. Reaction conditions: 100 μL, pH 13, 0.15 M H2O2.

CCCTGGGGGAGTAT-3′), C3 (5′-biotin-ACTTGGGGGAGTAT-3′), and A10 (5′-biotin-ACCTGGGGGCGTAT-3′), corresponding to the mutation of base G9, G8, G7, G6, A1, C3, and A10 (G to T, A to C, C to T) in the 14-mer aptamer fragment, respectively, instead of the ATP 14-mer aptamer fragment, were used to measure CL intensity. A scrambled sequence (S, 5′-biotin-TGGCGTGGACACGAAGATCAG-3′) was also examined as a reference. The dissociation constants of ATP binding mutated aptamer fragments G9, G8, G7, G6, A1, C3, and A10 sequences were measured. As shown in Figure 6a−f, the dissociation constants of G8, G7, G6, A1, C3, and A10 were 3.98, 4.04, 0.74, 1.03, 0.97, and 0.90 μM, respectively (Table S2). As shown in Figure 6g, the dissociation constant of the G9 sequence was too large to be obtained, indicating that G9 played a critical role in the folding process. S was a completely random-sequenced DNA, and its CL intensity was the lowest, fluctuating slightly with increasing ATP concentration. This result indicated that S could not bind ATP sufficiently (Figure 6h). The experimental results demonstrated the central role of G9 in the binding conformation and the

participation of G8, G7, G6, A1, C3, and A10 in the binding process as well. Molecular Dynamic Simulation of ATP-14mer Aptamer Fragment Complex. In order to confirm the binding site of the ATP-14-mer aptamer fragment complex, the binding mechanism of ATP and aptamer was studied by Molecular Dynamic (MD) simulation. Figure 7a−c represented the final binding conformation of the ATP binding14-mer aptamer complex. Two regions of the 14-mer fragment aptamer helped form the binding pocket that buried ATP: a multi-G region consisting of consecutive G6-A10 residues and 5′-terminal residues A1. The center of the ATP-binding site was characterized by an ATP·G9 recognition mismatch flanked by sheared A1·A10 and a normal Watson−Crick C3·G8 pair. The detailed simulation results are described in section S3 of the Supporting Information. According to experimental and simulation results, the binding site for ATP−aptamer interaction and the binding conformation of the ATP−aptamer complex are proposed in Figure 7d. The simulation results for the binding site strongly support the experimental results. 5563

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apt was 0.90 μM, which was comparable to the result of Williams and co-workers23 (Table S2). The CL intensity was also a linear function of the logarithm of TB concentration in the range of 1 pM to 10 nM with regression equation ICL = (4.61331 × 106) + (1.84726 × 105) × log C (R = 0.9961), as shown in Figure 8b (upper inset). The detection limit was (S/ N = 3) 0.32 pM, and the relative standard deviation of TB concentration was 1.7% at 1 nM (n = 7). As shown in Figure 8b (lower inset), the dissociation constant of TB-binding TBA was 66 nM, which was comparable to that measured by Bock and co-workers1 (Table S2). The effects of AVP and TB on the CL reaction between the ABEI−Au colloid and H2O2 were also studied (Figure 4). Similar to ATP, the CL intensity also increased with increasing AVP and TB concentration. These results imply that the proposed CL strategy could be suitable for multiple types of targets which could enhance ABEI−Au colloid-initiated CL. The CL enhancement of the luminol CL reactions by various organic compounds has been documented.41−44 Earlier studies showed that the CL enhancement of the luminol system was related to the generation of oxygen-related radicals such as OH•, O2•−, CO3•−, CO4•−, and other radical derivatives.41,45−48 It has been reported that ATP could enhance the CL of the luminol−H2O2−Fe2+ system, which was due to theh fact that ATP promoted hydroxyl radical production.47 Peptide AVP and protein TB are consisted of amino acids. They are carboxylcontaining molecules. It has been reported that phenolic compounds and amino acids containing −COO− groups could enhance the CL from the luminol−H2O2−Co2+ at higher pH.41 The CL enhancement might be due to the reaction of −COO− in their molecules with O2•− generated in the CL reaction to form −CO4•2−, which could react with luminol to facilitate the formation of luminol radicals. ABEI is an analogue of luminol. The CL enhancement by small molecule ATP, peptide AVP ,and protein TB of the ABEI−Au colloid−H2O2 CL reaction might be due to the fact that these compounds facilitate the formation of intermediate radicals, accelerating the ABEI−Au colloid−H2O2 CL reaction. Further work regarding the CL enhancement mechanism is under investigation. Compared with the reported methods8,17,18,21 for the measurement of the aptamer−target binding constant, the proposed CL strategy has some merits. For example, the proposed CL strategy only needs samples at the nanogram level, whereas NMR17,18 and ITC19 methods often need milligram-level sample amounts. Thus, the sensitivity of the CL method is 4 orders of magnitude higher than that of previously reported NMR and ITC methods. Moreover, the CL assay is label-free and avoids complicated labeling or separation procedures as well as expensive instrumentation, and it could rapidly measure the binding constant within 10 min (not including the incubation time). Thus, the proposed CL assay is sensitive, simple, fast, and low-cost. Besides, flow cytometry and fluorescence assay have technical limitations in labeling,24,25 inducing new steric hindrances in the configuration, whereas a small molecule can only lead to little change in signal in QCM21 and SPR22 assays. As such, they are difficult to be used for measuring small molecule−aptamer binding. Particularly, the proposed CL strategy can be used for measuring the binding constant of an aptamer with small molecules and macromolecules.

Figure 7. Illustration of ATP-binding pocket. (a) Surface representation of ATP-binding pocket. Red, oxygen; blue, nitrogen; white, carbon. (b) Left: Illustration of ATP·G9 recognition mismatch. Note that ATP is coplanar with G9, C2. Right: Illustration of two hydrogen bonds formed between the phosphate group of ATP and G6. (c) Illustration of sheared A1·A10 and normal Watson−Crick C3·G8 interactions. (d) Schematic illustration for the ATP-binding 14-mer aptamer fragment complex.

Generalization of the CL Strategy for Multiple Types of Targets. In order to investigate whether the CL strategy provides a general and effective method for measuring target− aptamer binding and target concentration, the CL measurement was successfully challenged with multiple types of targets, including nine-amino acid cyclic hormone peptide arginine vasopressin (AVP, Cys1-Tyr2-Phe3-Gln4-Asn5-Cys6-Pro7-Arg8Gly9-NH2) and its aptamer (V-apt, 5′-biotin-TCACGTGCATGATAGACGGCGAAGCCGTCGAGTTGCTGTGTGCCGATGCACGTGA-3′), as well as protein thrombin (TB) and its aptamer (TBA, 5′-biotin-GGTTGGTGTGGTTGG-3′), as shown in Figure 8. The CL intensity was a linear function of the logarithm of AVP concentration in the lower range of 0.1 nM to 1 μM with regression equation ICL = (1.98049 × 106) + (8.6401 × 104) × log C (R = 0.9944), as shown in Figure 8a (upper inset). The detection limit (S/N = 3) was 79 pM, and the relative standard deviation of AVP concentration was 2.4% at 0.1 μM (n = 7). As shown in Figure 8a (lower inset), the dissociation constant of AVP-binding V5564

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Figure 8. CL measurement of target−aptamer binding and target concentration using AVP and TB as targets. (a) Relationship between CL intensity and logarithm of AVP concentration. Upper inset: working curve for determination of AVP. Lower inset: measurement of dissociation constant of AVP-binding aptamer. (b) Relationship between CL intensity and logarithm of TB concentration. Upper inset: working curve for determination of TB. Lower inset: measurement of dissociation constant of TB-binding aptamer. Reaction conditions: 100 μL, pH 13, 0.15 M H2O2.

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CONCLUSION

binding of biologically important molecules to nucleic acids, having potential in fundamental research as well as in applications such as clinical diagnosis, pharmaceutical analysis, environmental monitoring, food safety assessment, etc. Besides, the proposed strategy might be generalized for studying the binding of biologically important molecules to biomacromolecules such as protein−small molecules binding and protein− macromolecule binding. Further work is under investigation. Finally, we have also discovered that the 14-mer aptamer fragment alone was able to selectively capture ATP, which was different from previous reports that two split DNA fragments from ATP aptamer were concurrently required to bind ATP. The binding constant, binding site, and binding conformation between the 14-mer aptamer fragment and ATP were obtained using such a novel CL strategy and MD simulation. Thus, this

We have described a general label-free chemiluminescence strategy for measuring aptamer−target binding and target concentration by taking small-molecule ATP, peptide AVP, and protein TB as model targets. It was found that all tested targets could enhance ABEI−Au colloid-initiated CL, suggesting the target universality of the approach. It was further shown that this strategy could achieve extremely high sensitivity with nanogram samples for measuring the target−aptamer binding constant. Moreover, the CL measurement could be rapidly performed without labeling or separation. And the methodologies for measuring both the aptamer−target binding and target concentration have rarely been reported. Therefore, with its high sensitivity, selectivity, simplicity, rapidity, and low cost, this CL strategy provides an effective tool for studying the 5565

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Analytical Chemistry

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work also demonstrates a good example for studying aptamer− target binding by virtue of a CL approach and MD simulation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text; S1. Optimization of CL detection conditions; S2. Binding fraction of 14-mer aptamer fragment for ATP; S3. Molecular Dynamic simulation of ATP14mer aptamer fragment complex. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-551-63606645. Fax: +86-551-63600730. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The support of this research by the National Natural Science Foundation of P. R. China (Grant Nos. 21173201, 21075115, 20625517, and 20573101) and the Fundamental Research Funds for the Central Universities (Grant No. WK2060190007) is gratefully acknowledged.

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REFERENCES

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dx.doi.org/10.1021/ac501061c | Anal. Chem. 2014, 86, 5559−5566