Highly Sensitive Detection of Protein with Aptamer-Based Target

Jan 8, 2012 - Hongquan Zhang , Feng Li , Brittany Dever , Xing-Fang Li , and X. .... Burcu Özay , Cara M. Robertus , Jackson L. Negri , Stephanie E. ...
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Highly Sensitive Detection of Protein with Aptamer-Based TargetTriggering Two-Stage Amplification Zhen-zhu Zhang and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China S Supporting Information *

ABSTRACT: Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. However, so far most detection methods rely on antibodybased assays and are usually laborious and time-consuming with poor sensitivity. Here, we develop a simple and sensitive method for the detection of a biomarker protein, plateletderived growth factor BB (PDGF-BB), based on aptamerbased target-triggering two-stage amplification. With the involvement of an aptamer-based probe and an exponential amplification reaction (EXPAR) template, our method combines strand displacement amplification (SDA) and EXPAR, transforming the probe conformational change induced by target binding into two-stage amplification and distinct fluorescence signal. This detection method exhibits excellent specificity and high sensitivity with a detection limit of 9.04 × 10−13 M and a detection range of more than 5 orders of magnitude, which is comparable with or even superior to most currently used approaches for PDGF-BB detection. Moreover, this detection method has significant advantages of isothermal conditions required, simple and rapid without multiple separation and washing steps, low-cost without the need of any labeled DNA probes. Furthermore, this method might be extended to sensitive detection of a variety of biomolecules whose aptamers undergo similar conformational changes.

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by chemical synthesis, which allows rapid preparation in large quantity and with excellent reproducibility; nucleic acid synthetic chemistry also facilitates the modification of aptamer sequences with fluorescent dyes and radiolabels.16−18 Recently, aptamer-based fluorescent,19 electrochemical,20 and surfaceenhanced Raman spectroscopy (SERS) methods21 have been developed for protein detection. While substantial progress has been accomplished, there still has been a major challenge that hinders biomarker detection; i.e., some candidate biomarkers exist at extremely low concentrations, and the routine aptameric methods are not sensitive enough to detect them.22−25 Alternatively, amplification approaches are employed in aptamer-based assay, and several impressive works have been reported, such as polymerase chain reaction (PCR) assay based on proximitydependent DNA ligation26 and micromagnetic bead-based aptameric PCR.27 Although these assays in principle offer extremely high sensitivity and wide quantitative dynamic range, they are usually too complicated and time-consuming for practical applications. Several groups have developed new approaches based on aptamer-ligand recognition-induced

roteins are ubiquitous and essential in all living organisms. The recognition and quantification of disease-marker proteins, especially those associated with cancers, are of particular importance in the fundamental research and practical applications such as medical diagnosis, prevention, and treatment.1 Consequently, methods that allow rapid, simple, sensitive, selective, and cost-effective detection of proteins play critical roles in new disease-marker discovery and their function studies.2,3 In general, antibody-based assay is a versatile and powerful tool for specific antigen detection.4 However, it is faced with the considerable challenges of limited dynamic range,5,6 insufficient sensitivity,7 and long assay times with the involvement of multiple washing steps.8 As complements to the antibody-based assay, molecular probes based on aptamerprotein recognition have gained increasing attention recently.9−11 Aptamers are artificial, short, single-stranded oligonucleotides isolated from random-sequence nucleic acid libraries through an in vitro selection process called SELEX (selective evolution of ligands by exponential enrichment).12−14 Aptamers typically assume defined, compact secondary and tertiary structures, and demonstrate high affinity (Kd values in the nanomolar range) and good selectivity for their targets.15,16 Importantly, aptamers are more stable than proteins under a wide range of conditions, and can be repeatedly used without losing the binding capabilities.16 In addition, aptamers can be routinely prepared © 2012 American Chemical Society

Received: November 2, 2011 Accepted: January 8, 2012 Published: January 8, 2012 1623

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rolling circle amplification (RCA),28,29 but few have achieved the sensitivity of PCR. Willner’s group demonstrates a strand displacement amplification (SDA) based on target recognitioninduced conformational changes.30 The target-bound-aptamer machine can be activated with the help of polymerase and nicking enzyme, continuously yielding short oligonucleotides which are detected by the molecular beacon. This assay is easy to operate, whereas the disadvantage is high cost with the requirement of double-labeled molecular beacon which is difficult to optimize and synthesize. In the present study, we develop a novel detection method, which involves aptamer-based target-triggering SDA and exponential amplification reaction (EXPAR), for sensitive detection of protein. EXPAR, which was devised by Galas and co-workers to detect short oligonucleotides,31 is a combination of polymerase strand extension and single-strand nicking. In contrast to other amplification methods, EXPAR has distinct advantages of its isothermal nature, high amplification efficiency, and rapid amplification kinetics with 106−109-fold amplification of short oligonucleotides in minutes.31 Plateletderived growth factor B-chain (PDGF-BB), an important protein for cell transformation, tumor growth, and progression,32,33 is selected as the model protein. In the presence of protein, a structure-switching probe designed from the original aptamer can be amplified and nicked, generating short oligonucleotides. These short oligonucleotides are the triggers of EXPAR, converting single-stranded EXPAR templates to double-stranded DNAs (dsDNAs) which can be simply detected by fluorescent dye SYBR Green I. Due to the high amplification efficiency, a detection limit of 9.04 × 10−13 M and a dynamic range of more than 5 orders of magnitude might be achieved. This detection method is comparable with or even superior to most currently used approaches for protein detection. In addition, since a variety of aptamers (original or adapted sequences) undergo similar conformational changes upon target binding,16 this detection method might be further extended to sensitive detection of other proteins and small molecules.

PeproTech (Rocky Hill, NJ) and reconstituted in 4 mM HCl with 0.1% bovine serum albumin (BSA). Fetal bovine serum (FBS) and BSA were purchased from Sigma-Aldrich (St. Louis, MO). All reagents were prepared and handled according to the supplier’s specifications. Prior to use, all DNA and PDGF proteins were diluted with PBSM buffer (137 mM NaCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 2.7 mM KCl, 1 mM MgCl2). The deoxynucleotide triphosphates (dNTPs) were obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Klenow fragment polymerase (3′→5′ exo-, KF polymerase) and the nicking endonuclease Nt.BbvCI were purchased from New England Biolabs Ltd. SYBR Green I (20× stock solution in dimethyl sulfoxide, 20 μg/mL) was purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). Low molecular weight DNA marker (SM0303) was purchased from MBI Fermentas (Canada). Detection of PDGF-BB. A 1 μL sample of PDGF-BB solution was mixed with 1 μL of 10 nM probe system, and the aptamer-target binding was allowed to proceed at 37 °C for 15 min. In the meantime, the EXPAR reaction mixtures were prepared separately on ice as part A and part B. Part A consisted of NEB buffer 2, the amplification template, and dNTPs. Part B consisted of the nicking endonuclease Nt.BbvCI, KF polymerase in 1× NEB buffer 2, and SYBR Green I. Parts A and B were added immediately to the incubation solution containing the probe and protein before being placed in the real-time PCR system. The EXPAR was performed at 37 °C in a volume of 10 μL solution containing the amplification template (0.05 μM), dNTPs (250 μM), Nt.BbvCI (0.2 U/μL), KF polymerase (0.1 U/μL), SYBR Green I (0.4 μg/mL), 1× NEB buffer 2 (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9). The real-time fluorescence measurements were performed with a Roche LightCycler 480 (Switzerland), and the fluorescence intensity was monitored at intervals of 30 s.

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RESULTS AND DISCUSSION Scheme for Aptamer-Based Target-Triggering TwoStage Amplification. Our strategy for protein analysis is illustrated in Scheme 1. The aptamer-based probe consists of three regions: Region I includes the aptamer region for PDGFBB. Region II is the “heart” of the probe, wherein a recognition site for Nt.BbvCI is generated upon the formation of a dsDNA. Region III is complementary to a short oligonucleotide (the product of SDA) which is the trigger of EXPAR. The probe is blocked by a blocker DNA (3′ end with 3 base noncomplementary to the probe) to prevent it from uncontrolled folding into an active configuration in the absence of targets.30 The EXPAR template consists of two copies of the region III separated by the region II. The addition of PDGF-BB separates the blocker DNA from the probe, making the probe fold into a close configuration. The 3′ end of the probe can function as a primer to initiate an extension reaction in the presence of Klenow fragment DNA polymerase and dNTPs, yielding a dsDNA with the recognition site for Nt.BbvC I. Subsequent single-stranded nicking generates a new replication site for the polymerase, and the SDA replication can displace the downstream oligonucleotides. The SDA cycle includes polymerization, nicking, and subsequent release of the oligonucleotides. The linear SDA reaction can be turned into an exponential amplification with the short oligonucleotides as the triggers of EXPAR. After hybridization of the trigger

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EXPERIMENTAL SECTION Materials. Oligonucleotides used in this work were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and their sequences were listed in Table 1. Table 1. Sequence of Probe and Template Oligonucleotidesa note

sequence (5′−3′)

probe with 5 bp stem probe with 7 bp stem probe with 9 bp stem

TAG GTG AAC GTG GAG CTG AGG TTT CAC ACA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG T TAG GTG AAC GTG GAG CTG AGG TTT CAC ACA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG TGT TAG GTG AAC GTG GAG CTG AGG TTT CAC ACA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG TGT GA TAG GTG AAC GTG GAG CTG AGG GCT AGG TGA ACG TGG AGC TGA-P CCT GTG TGA AAC TCT

EXPAR template blocker DNA a

The boldfaced regions of the probe indicate anti-PDGF-BB aptamer. P in EXPAR template represents phosphate at the 3′ end.

The DNA stock solutions were prepared with the buffer containing 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, and 2 mM MgCl2. The dimeric isoforms of PDGF proteins (PDGFAA, PDGF-AB, and PDGF-BB) were purchased from 1624

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Scheme 1. Scheme for Sensitive Detection of PDGF-BB with Aptamer-Based Target-Triggering Two-Stage Amplificationa

a This involves three principal steps: (1) target-aptamer binding and blocker DNA dissociation, (2) linear strand displacement amplification, and (3) exponential amplification reaction. PDGF-BB acts as a kind of “catalyst” to trigger the conformational change of otherwise inactive probe into an active structure, generating short oligonucleotides linearly, and these oligonucleotides can trigger the exponential amplification reaction whose products can be detected by SYBR Green I.

oligonucleotide with the 3′ end of the EXPAR template, it can be extended by the polymerase, and the resulting dsDNA can be recognized and nicked by the nicking enzyme, recreating a primer template for another elongation. As a result, hybridization, extension, nicking, and strand displacement amplification can be repeated continuously, leading to an exponential amplification and consequently a large amount of dsDNAs. In this study, SYBR Green I is utilized as the fluorescent dye for the detection due to its high specificity for dsDNAs.34 The amplification scheme described here has significant advantages of isothermal conditions required, rapid and simple without multiple separation and washing steps, label-free, and low-cost. Direct evidence for the activation of the probe system by PDGF-BB and subsequent two-stage amplification is provided by gel-electrophoresis analysis with a 12% nondenaturating PAGE gels (see Supporting Information, Figure S1). Real-Time Fluorescence Measurement. Real-time fluorescence measurement with SYBR Green I as the fluorescent dye is used to monitor the activation of the probe system by PDGF-BB and the subsequent amplification reaction. As shown in Figure 1, the control reaction without the EXPAR template does not show any detectable amplification in the fluorescence measurement (Figure 1, curve a), because the fluorescence dye SYBR Green I exhibits negligible fluorescence in the presence of short single-stranded oligonucleotides (products of SDA reaction).35 The real-time fluorescence intensity increases in a sigmoidal fashion in the presence of 1 nM PDGF-BB (Figure 1, curve b) as the amplification templates are converted from single-stranded to partially or completely double-stranded DNAs, eventually reaching a plateau once all the singlestranded templates have been converted into dsDNAs. This is consistent with previous reports of EXPAR.36 The point of inflection (POI, the time corresponding to the maximum slope in the fluorescence curve) is used for quantitative detection of target proteins.34,36 It should be noted that the negative control without PDGF-BB, which contains all components of the

Figure 1. Real-time fluorescence monitoring of the amplification reaction. The data correspond to the relative fluorescence of control reaction (a) without EXPAR template, and two-stage amplification in the (b) presence of PDGF-BB or in the (c) absence of PDGF-BB. The probe concentration is 1 nM, the template concentration is 0.1 μM, and PDGF-BB concentration is 1 nM.

detection system, displays nonspecific background amplification as well; however, it takes more time to reach a plateau (Figure 1, curve c). This nonspecific background amplification might be attributed to the following two factors: (1) templated but unprimed DNA synthesis, which makes the polymerase bind to a single-stranded EXPAR template and synthesize DNA complementary to the template in an unprimed fashion, or prime in an unconventional manner;37 (2) extension of the template at the 3′-end by the polymerase, which forms template dimers similar to “primer-dimer” type nonspecific background amplification observed in PCR.36 Since the nonspecific background amplification exhibits a high POI value, it does not interfere with the specific amplification in the presence of target proteins. Optimization of Aptamer Probes. The original sequence of PDGF-BB aptamer identified is a 39-mer sequence with an 1625

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have demonstrated that background amplification by thermophilic polymerases in the absence of any templating or priming DNA strands is accelerated in the presence of restriction endonucleases or nicking endonucleases.37,40 Although the precise mechanism for the enzymatic interaction, cooperation, or competition with one another remains unclear, the reaction might be simply modulated by changing the ratio of the enzymes.31,41 With a fixed concentration (0.1 U/μL) of Klenow fragment polymerase as reported previously,30 the real-time fluorescence curve produced by 1 nM PDGF-BB is measured in the presence of 0.1 U/μL, 0.2 U/μL, 0.3 U/μL, and 0.4 U/μL Nt.BbvCI nicking endonuclease (see Supporting Information, Figure S3), respectively. The maximum interval of the POI values between PDGF-BB and the negative control without PDGF-BB is obtained when the Nt.BbvCI concentration is 0.2 U/μL. Therefore, 0.2 U/μL Nt.BbvCI nicking endonuclease is selected in the following research. Optimization of EXPAR Template Concentration. The concentration of EXPAR template has a crucial effect on the specificity and efficiency of the amplification. On one hand, a high concentration of EXPAR template might lead to high background amplification due to the fact that the nonspecific background amplification involves templated but unprimed DNA synthesis as well as the formation of template dimers similar to “primer-dimer” observed in PCR.36,37 On the other hand, low concentration of EXPAR template might adversely affect the amplification efficiency because the amplification efficiency of EXPAR depends on the ability of short oligonucleotides to hybridize with the template.31,34 Therefore, the concentration of EXPAR template should be optimized carefully. As shown in Figure S4 (see the Supporting Information), with the increase of template concentration, the fluorescence intensity increases in the presence of PDGF-BB; however, the fluorescence intensity increases in the negative control without PDGF-BB as well. When the concentration of EXPAR template is 0.05 μM, the interval of the POI values between PDGF-BB and the negative control reaches the maximum. Therefore, 0.05 μM EXPAR template is selected in the following assay. Improved Sensitivity for PDGF-BB Detection. To demonstrate the improved sensitivity of the current assay, we detect PDGF-BB at various concentrations by real time fluorescence measurement. As shown in Figure 3A, the POI decreases monotonically with the increase of protein concentration, indicating that the concentration of target protein is directly proportional to the copy number of active probe.27,34 Moreover, a large dynamic range from 640 fM to 50 nM is obtained. In logarithmic scales, the POI exhibits a linear correlation to the concentration of target protein through a 5decade range from 3.2 pM to 50 nM (Figure 3B). The regression equation is Y = −66.132 − 10.83 log10 C with a correlation coefficient of 0.9925, where Y and C represent the POI and the PDGF-BB concentration, respectively. Notably, the dynamic range of the current assay is about 3 orders of magnitude larger than the RCA-based electrochemical methods,28 and 2 orders of magnitude larger than the aptameric fluorescent methods for PDGF-BB detection.42,43 To obtain the detection limit, the standard deviation value of the signals from the negative control is multiplied by 3 and subtracted from the mean negative control signal to reach a limit of detection (LOD)44 of 9.04 × 10−13 M. This detection limit is 103−104-fold lower than those obtained in real-time RCA29 and RCA-based electrochemical methods.28 This low

open secondary structure in the absence of PDGF-BB, and a close conformation of a three-way helix junction where the 3′ and 5′ ends of the aptamer form a stem in the presence of PDGF-BB.38 However, the theoretical calculations indicate that a significant fraction of aptamer will be in close conformation even in the absence of target proteins,16,39 leading to nonspecific amplification. To prevent the probe conformational change in the absence of target proteins, we add a blocker DNA with 3 bases noncomplementary to the probe in the 3′ end (Table 1). Our experiment demonstrates that the blocker DNA can significantly reduce the nonspecific background amplification and improve the specificity of amplification (see Supporting Information, Figure S2). The length of stem is important for the aptamer-based assay. The efficiency of the replication process strongly depends on the ability of the 3′ end of the probe to hybridize with its outer sequences and the stability of the formed stem.16,28 The lengthening of the stem facilitates the replacement of block DNA and the formation of folded structure, and in turn triggers the amplification. However, when the stem of probe is too long, the 3′ end of the probe can replace the blocker DNA and form a double-stranded stem even in the absence of target proteins.30,39 To optimize the length of the outer stem, three probes with 5, 7, and 9 bp stems are designed and tested, respectively. As shown in Figure 2, the POI decreases

Figure 2. Effect of stem length of the probe on the amplification. The real-time fluorescence curves of amplification in the presence (○) and in the absence (●) of 1 nM PDGF-BB. The stem length of probes is 5 bp, 7 bp, and 9 bp, respectively. The concentration of each probe is 1 nM. Error bars show the standard deviation of three experiments.

monotonically with the increase of stem length, and both probes with 5 bp and 9 bp stems exhibit less discrimination efficiency against PDGF-BB as compared to the probe with 7 bp stem. Therefore, the probe with 7 bp stem is selected in the following assay due to its good performance in activating SDA and EXPAR in the presence of PDGF-BB, but remaining inactive in the absence of PDGF-BB. Optimization of Nicking Endonucleases Concentration. The detection sensitivity of protein assay based on nucleic acid amplification is mainly limited by nonspecific background amplification, which is influenced by the assay conditions such as concentrations of template, polymerase, and nicking endonuclease.31,36 The balance between the nicking enzyme and the DNA polymerase is complex. Some literature studies 1626

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Figure 4. Variance of the POI value as a function of PDGF-BB concentration in FBS. Inset: Linear relationship between the POI and the logarithm of PDGF-BB concentration in FBS. Final concentrations: [EXPAR template] = 0.05 μM, [each dNTP] = 250 μM, [Nt.BbvCI] = 0.2 U/μL, [Klenow fragment (exo-) DNA polymerase] = 0.1 U/μL, [SYBR Green I] = 0.4 μg/mL. Error bars show the standard deviation of three experiments.

is much simpler, cost-effective, and rapid with the requirement of less than 1 h, whereas ELISA assay usually requires more than 5 h. Detection Specificity. For the aptamer-based assays, the detection specificity is essentially determined by the intrinsic properties of aptamer. Although the original PDGF aptamer has been proved to specifically recognize its target molecule,38 the probe used in the current assay has been adapted by lengthening the sequence. This possibly leads to a change (deterioration or improvement) in its target recognition ability.46,47 In order to evaluate the detection specificity of current assay, the ΔPOI of a particular sample is measured (the ΔPOI is defined as the difference in the POI value between a particular sample with the protein and the negative control without the protein). The dimeric isoforms of PDGF proteins (PDGF-AA, PDGF-AB, and PDGF-BB) with the same concentration are tested under identical conditions. Human serum albumin (HSA) (0.005%) is also tested for the control groups. As shown in Figure 5, the ΔPOI of HSA is 2, and the ΔPOI of PDGF-AA is 132. In contrast, the ΔPOI of PDGF-BB is 1014, indicating the high specificity of the current assay for PDGF-BB detection. The significant difference in the ΔPOI between PDGF-BB and PDGF-AA is in agreement with the literature reports that this aptamer exhibits 500-fold lower affinity for the PDGF A-chain than for the PDGF B-chain.26,27 In addition, the ΔPOI of PDGF-BB and that of PDGF-AB are remarkably similar in magnitude; this is consistent with the fact that the dissociation constant (Kd) of the aptamer for PDGFBB is nearly identical to that of the aptamer for PDGF-AB.27 Moreover, the detection specificity of the current assay compares favorably with the reported light-switching excimer aptamer16 and RCA-based electrochemical methods28 for PDGF-BB detection.

Figure 3. Sensitive detection of PDGF-BB. (A) Real-time fluorescence curves obtained from the amplification triggered by PDGF-BB at various concentrations. (B) Variance of the POI value as a function of PDGF-BB concentration. Inset: Linear relationship between the POI value and the logarithm of PDGF-BB concentration. Final concentrations: [EXPAR template] = 0.05 μM, [each dNTP] = 250 μM, [Nt.BbvCI] = 0.2 U/μL, [Klenow fragment (exo-) DNA polymerase] = 0.1 U/μL, [SYBR Green I] = 0.4 μg/mL. Error bars show the standard deviation of three experiments.

detection limit might be attributed to the high amplification efficiency of two-stage amplification. Quantitative Detection of PDGF-BB in FBS. To be useful in the bioassays, the developed detection method should be able to tolerate the interference from the biological samples.16,27 Therefore, we prepared a series of samples by adding different concentrations of PDGF-BB to fetal bovine serum (FBS). As shown in Figure 4, we obtain a large dynamic range from 16 pM to 50 nM, which spans 4 orders of magnitude. The regression equation is Y = −118.06 − 19.142 log10 C with a correlation coefficient of 0.9892, where Y and C represent the POI and the PDGF-BB concentration, respectively. A limit of detection of 9.24 × 10−12 M is obtained, which is somewhat higher than that in pure buffer. This might result from the reduction of enzyme activity or aptamer-protein binding affinity in FBS.16 It should be noted that our assay is more sensitive than the reported light-switching excimer aptamer16 with a LOD of 2.5 nM and the aptamer-based electrochemical detection method20 with an LOD of 50 pM for real sample measurement. ELISA is the most standard method currently used for the detection of PDGF-BB. Previous research demonstrated that ELISA assay for PDGF-BB detection spanned 4 orders of magnitude (from 10 pM to 10 nM) with an LOD at the picomolar level.26,45 Our detection method compares favorably with ELISA for real sample detection. In addition, our method

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CONCLUSIONS In summary, we have developed a simple and sensitive method for the detection of PDGF-BB based on aptamer-based targettriggering two-stage amplification. This method utilizes an 1627

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the Innovation Team Project of Guangdong Province, the Natural Science Foundation of Shenzhen City (Grant JC201005270327A), and the Award for the Hundred Talent Program of the Chinese Academy of Science.

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Figure 5. Detection specificity measured by the ΔPOI. The samples tested are 10 nM PDGF-BB (BB), 10 nM PDGF-AB (AB), 10 nM PDGF-AA (AA), 0.005% HSA, and a negative control without the protein. The ΔPOI of the negative control is defined as 0. Error bars show the standard deviation of three experiments.

aptamer-based probe and an EXPAR template to transform the probe conformational change induced by target binding into two-stage amplification and distinct fluorescent signal. Due to the high affinity and specificity of aptamers for proteins38 and the high amplification efficiency of both SDA and EXPAR,30,31,34 this method exhibits excellent specificity and high sensitivity with a detection limit of 9.04 × 10−13 M and a detection range of more than 5 orders of magnitude. Moreover, it functions well in FBS, holding great potential for further applications in clinical diagnosis. In comparison with aptameric RCA-based electrochemical method28 with the requirement of more than 5 h and micromagnetic bead-based aptameric PCR method27 with the involvement of many separation steps, our method is much simple and rapid (less than 1 h) with an isothermal condition but without the need of any thermal cycling, washing, and separation steps. Moreover, in contrast to the light-switching excimer16 and proximity-dependent DNA ligation assays,26 this method is much more cost-effective with SYBR Green I as the fluorescent dye but without the need of any labeled DNA probes. In addition, since a variety of aptamers for cocaine,48 THR,18,49 and HIV1 Tat protein17 undergo similar conformational changes upon target binding, this method might be further extended to sensitive detection of other proteins and small molecules.

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ASSOCIATED CONTENT

S Supporting Information *

Gel electrophoresis details and Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected].

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grants 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant 21075129), the Knowledge Innovation Project of the Chinese Academy of Science (Grant KGCX2-YW-130), the Award for 1628

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on January 24, 2012. Additional text corrections were added to the figure captions, and the corrected version was published on January 27, 2012.

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dx.doi.org/10.1021/ac2029002 | Anal. Chem. 2012, 84, 1623−1629