Turn-On Aptameric System for Simple and Selective Detection of

ARTICLE pubs.acs.org/ac

Turn-On Aptameric System for Simple and Selective Detection of Protein via Base Stacking-Dependent DNA Hybridization Event Sheng Cai, Choiwan Lau, and Jianzhong Lu* School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China

bS Supporting Information ABSTRACT: Base stacking is employed in an entirely new type of sensing platform for the simple and robust detection of protein. Only in the presence of protein, the aptamer DNA can hybridize stably with the capture DNA to form a stem-loop structure due to the enhancement of base stacking. This leads to a strong chemiluminescence emission for simple protein detection. With the use of a platelet-derived growth factor as a model, a fM detection limit was obtained with a dynamic range that spanned 4 orders of magnitude. Upon modification, the approach presented herein was also extended to detect other types of targets including Hg2+ ion and adenosine and also other types of labels such as fluorescence nanogold. We believe such advancements will represent a significant step toward improved diagnostics and more personalized medical treatment and environmental monitoring.

E

nzyme-linked immunosorbent assay (ELISA) is a standard approach for detection of protein biomarkers directly from blood. Unfortunately, this assay can require a lengthy development period for specific antibodies1 and long assay times involving multiple washing steps.2 While antibody-based immunoassay methodologies are still routinely used for protein detection, aptamers have become attractive alternatives to antibodies for use as molecular recognition elements in biosensor applications due to the enormous versatility of nucleic acid components in labeling, immobilization, signaling, and amplification.38 Numerous aptasensors for protein detection have been developed with mechanical, optical, or electronic readouts, as protein quantification is critical for basic research and clinical applications.912 Although these aptamer methods have demonstrated specific protein detection with useful working ranges, the development of a simple and robust assay with improved analytical properties is important for use in practical applications (see Scheme 1). Herein, we report an innovative protein detection technology to improve the simplicity and robustness of the aptameric system based on the formation of a stem-loop structure and enhancement of base stacking. Our new technique encompasses a design strategy whereby capture and aptamer probes that do not hybridize to each other are made to anneal to each other in the presence of a target protein via the formation of a proteinaptamer complex, leading to the formation of a stem-loop structure and enhancement of base stacking. Specifically, the aptamer probe has an additional tethered 30 -biotin-labeled eightnucleotide tail sequence that is complementary to a surfacetethered capture DNA with a predesigned low melting temperature (usually PDGFAB > PDGF-AA.23 The CL response of IgG, IgA, IgM, and interferon was also examined at 500 pM or 5 nM concentrations. The ratios of CL intensities of the seven antigens were 100:11.2:2.2:1.7:1.4:1.7:1.5:1.3, respectively (Figure 1). Hence, good selectivity was obtained using this new CL protein aptasensor. These results demonstrate that neither nonspecific adsorption of non-PDGF aptamer nor nonspecific aptamer binding formed on the surface of the 96-well surface blocked with BSA. Note that the original PDGF aptamer needs to be redesigned for the sensitive and selective detection of the target protein. The consensus secondary structure motif of the PDGF aptamer is a three-way helix junction with a three-nucleotide loop at the branch point, and the helix junction domain represents the core of the structural motif required for high-affinity binding. Our aptamer was designed to include the aptamer sequence and a 50 -biotin-labeled eight-nucleotide tail sequence that was complementary to a surface-tethered capture DNA. Figure 2 shows 5846

dx.doi.org/10.1021/ac201054m |Anal. Chem. 2011, 83, 5844–5850

Analytical Chemistry

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Figure 2. CL intensity (red) and CL ratio (blue) vs different aptamer DNAs. Experimental conditions: capture A and biotinylated aptamer DNA were used at 100 and 6 pmol, respectively, and PDGF-BB was 500 pM; streptavidin-HRP 6.67 ng (0-bp, aptamer D; 2-bp, aptamer E; 4-bp, aptamer A; 6-bp, aptamer F; 8-bp, aptamer G). The detection procedure was performed as described in the Experimental Section.

Figure 3. CL intensity vs different capture-aptamer pairs. Experimental conditions: capture probe (100 pmol) and biotinylated aptamer DNA (6 pmol) (B-B, capture B-aptamer B; B-H, capture B-aptamer H; A-A, capture A-aptamer A; A-G, capture A-aptamer G; C-C, capture C-aptamer C); PDGF-BB 500 pM (blue) and 0 pM (red); streptavidin-HRP 6.67 ng. The actual CL intensity for C-C was multiplied by 0.1. The detection procedure was performed as described in the Experimental Section.

the secondary structures of five aptamer sequences and CL intensities in the absence and presence of PDGF-BB. The number of base pairings at the 30 terminal of the aptamer greatly affected our sensor performance. More than two base pairings are required to stabilize the aptamer-PDGF stereostructure. However, blank signal in the absence of PDGF-BB also increases with the increase of base pairing at the 30 terminal of the aptamer. Thus, we chose an aptamer with four base pairings in the subsequent experiments. The number of base pairings between the capture and aptamer probe is also a key parameter for a successful assay system for PDGF-BB. Figure 3 shows a comparison between three capture

probes and five aptamer probes. Note that the cumulative base pairing and stacking could not stabilize the hybridization between the capture probe B and the aptamer B or H, leading to a very low CL intensity even in the presence of PDGF-BB. In contrast, the capture probe with 12 base pairings can stably hybridize with the aptamer, resulting in a high CL intensity even in the absence of target protein, and thus the addition of the target protein only causes a slight increase in CL intensity. Capture A with eight base pairings was found to be the best one and then selected for use in further studies. Overall, the lengths of both the capture and aptamer probes play key roles in this innovative protein detection technology via base-stacking-dependent surface-hybridization events. In the absence of target PDGF-BB, the strand length is only eight base pairings between capture and aptamer probes. As a result, the DNA double helix is unstable and the CL intensity is therefore very low (Figure S1 in the Supporting Information). Upon the introduction of protein, the aptamer is triggered to make a structure, switching to the formation of stem-loop structure and coaxial stacking at the 50 -end of the capture probe brings additional stability and efficiency to the hybridization as a result of increased association and decreased dissociation.24,25 Thus, the cumulative base pairing and stacking between adjacent bases stabilizes the hybridization between capture and aptamer probes, leading to a strong CL emission. We also tested the detection when the gap between the capture and aptamer probes was one or four nucleotides (Figure S2 in the Supporting Information). The CL intensity was found to decrease sharply with an increasing number of gap nucleotides. The hybridization was destabilized because a 1 or 4 nucleotide gap was generated between the capture and aptamer DNA. This interruption in the coaxial stacking results in destabilization of the three-component hybridization, thus decreasing 5847



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Figure 4. Calibration curve for PDGF-BB. Experimental conditions: capture A (100 pmol) and aptamer A (6 pmol); streptavidin-HRP was 6.67 ng. The detection procedure was performed as described in the Experimental Section. The insert photo is the CL image.

Table 2. Comparison of Sensitivity for Different PDGF Assay Methods analytical method

label

detection limit

electrochemical detection

label-free

50 fM26

electrochemical detection electrochemical detection

ferrocene alkaline phosphatase (ALP)

40 fM11 10 fM28


label-free

63 pM31


gold nanoparticles

10 fM32

fluorescence anisotropy

fluorescein

0.22 nM33

fluorescence detection

gold nanoparticles

10 fM34


fluorescein

110 pM35


gold nanoparticles

8 pM36

diffractometric detection Raman spectroscopy

magnetic microbeads label-free

10 pM37 1 μM38

PCR

label-free

333 pM39

micromagnetic aptamer PCR label-free

63 fM30

capillary electrophoresis

label-free

50 pM40

CL technique (this work)

HRP

10 fM

the CL signal. These gap experiments further confirm that the origin of the enhancement of the thermodynamic stability of adjacent helixes upon the introduction of protein can result from coaxial stacking. As such, we reasoned that the recognition of the target protein by the aptamer sequence was a prerequisite for the efficient hybridization of the aptamer probe with the surface-attached capture strand. Moreover, no obvious CL signal was observed in the control experiments by using noncomplementary capture X instead of capture A, indicating that surface hybridization was essential in signaling target protein. Actually, the recognition of target protein by the aptamer probe and hybridization of the aptamer probe with immobilized strands represent two key steps for the highly selective detection of target protein.

The quantitative behavior of the assay under the optimized experimental conditions was assessed by monitoring the dependence of the CL intensity on the target PDGF-BB concentration. As shown in Figure 4, CL intensity was proportional to the concentration of PDGF-BB. The linear curve fitted a regression equation of Lg I = 0.6269 Lg C + 3.7048 within a range from 50 fM to 500 pM with a correlation coefficient of R2 = 0.9827, where I is the net CL intensity (the signal minus the noise) and C is the concentration of target PDGF-BB. The detection limit was estimated to be 10 fM at a signal-to-noise ratio of 3, with a large dynamic range that spanned approximately 4 orders of magnitude (50 fM to 500 pM). Taking the sample volume into account, we detected approximately 1 amol of protein molecules. Note that this method is similar to ELISA in terms of target binding and read-out. However, two key factors possibly offer significant advantages that lead to dramatic sensitivity improvement. First, the surface of 96-well plate is covered with many capture probes with negatively charged phosphate groups, and this hydrophilic surface effectively inhibits the nonspecific approach and adsorption of PDGF-BB and SA-HRP, leading to that the background signal is much lower than that by ELISA. Second, this present method is much simpler than ELISA, nonspecific adsorption is comparatively low and thus the ratio of signal/noise is much higher. The sensitivity of our assay compares favorably with previous efforts using the PDGF-BB aptamer (Table 2); for example, a lightswitching excimer probe offered a detection limit of 4 pM,4 while an electrochemical approach yielded a detection limit of 50 pM.26 By using a detection scheme that incorporates real-time PCR, Yang and Ellington detected PDGF-BB with a LOD of 12.8 pM,27 and lowfemtomolar detection limits were generally obtained with a proximity ligation assay and a rolling-circle amplification-based method using an antibody-aptamer pair.11,28,29 In addition, Csordas et al.30 combined microfluidic magnetic sample preparation with the aptamer-based molecular recognition and quantitative PCR to achieve detection of PDGF-BB at concentrations of 50 fM. A series 5848



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this technique provides a novel method for simple, fast, and convenient point-of-care diagnostics in the field monitoring of proteins and also toxic metal ion, etc.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Figure 5. Relationship between the proposed method and the ELISA for PDGF-BB. Experimental conditions: capture A (100 pmol) and aptamer A (6 pmol); streptavidin-HRP was 6.67 ng. The detection procedure was performed as described in the Experimental Section.

of six repetitive measurements of 1, 100, and 500 pM PDGF-BB were used for estimating the precision, yielding a relative standard deviation of 1.61%, 1.50%, and 5.77%, respectively. To demonstrate the applicability of the proposed protein detection technology to clinical diagnosis, 20 human serum samples from normal individuals and patients were taken and divided into two aliquots, one for our method and one for a human PDGF ELISA Kit as a reference method. As can be seen from Figure 5, the PDGF-BB concentrations obtained by the presented method are in good agreement with those determined by the ELISA method. The correlation was calculated with a linear least-squares method, and the 95% confidence intervals of the slope and intercept were 0.90977 to 1.08483 and 1.76377 to 1.69945 for PDGF-BB, respectively. The confidence intervals for the slope and intercept values include the unit and zero values, respectively; thus, the results indeed showed no significant difference between the conventional assay and the proposed one, as confirmed by the Student t test (P > 0.05). Assays on serum from normal individuals and patients clearly indicated that this assay would be suitable for the routine assay of clinical samples.

’ CONCLUSIONS In conclusion, we developed a novel sensing platform for the simple and robust detection of protein based on a base stackingdependent surface-hybridization assay. The method required the recognition of target protein by an aptamer probe to promote their efficient annealing with 96-well surface-attached capture DNA. Note also that previous designs of highly sensitive protein aptasensors generally require the immobilization of corresponding antibody or aptamer on a surface for capture of the aptamer functionalized label, and thus those aptasensors will not work well if the binding of antibody to the target protein interferes with the binding of aptamer to the protein. By contrast, the method described in this work is simple, rapid, economic, sensitive, specific, and appears well suited for the direct detection of proteins. Additionally, the approach presented herein could also be extended to detect other types of targets including Hg2+ ion (Scheme S2 and Figure S8 in the Supporting Information) and adenosine (Scheme S1 and Figure S7 in the Supporting Information) and also other types of labels such as fluorescence nanogold (Figure S6 in the Supporting Information). Overall,

’ ACKNOWLEDGMENT We acknowledge financial support from the National Drug Innovative Program (Grant 2009ZX09301-011), National Natural Science Foundation of China (Grant 20975026), and the Research Fund for the Doctoral Program of Higher Education (Grant 20090071110056). ’ REFERENCES (1) Baker, K. N.; Rendall, M. H.; Patel, A.; Boyd, P.; Hoare, M.; Freedman, R. B.; James, D. C. Trends Biotechnol. 2002, 20, 149–156. (2) Lam, M. T.; Wan, Q. H.; Boulet, C. A.; Le, X. C. J. Chromatogr. 1999, 853, 545–553. (3) Jayasena, S. D. Clin. Chem. 1999, 45, 1628–1650. (4) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17278–17283. (5) Zhu, Z.; Wu, C. C.; Liu, H. P.; Zou, Y.; Zhang, X. L.; Kang, H. Z.; Yang, C. J.; Tan, W. H. Angew. Chem., Int. Ed. 2010, 49, 1052–1056. (6) Zuo, X. L.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131, 6944–6945. (7) Zuo, X. L.; Song, S. P.; Zhang, J.; Pan, D.; Wang, L. H.; Fan, C. H. J. Am. Chem. Soc. 2007, 129, 1042–1043. (8) Zayats, M.; Huang, Y.; Gill, R.; Ma, C. A.; Willner, I. J. Am. Chem. Soc. 2006, 128, 13666–13667. (9) Hartwell, L.; Mankoff, D.; Paulovich, A.; Ramsey, S.; Swisher, E. Nat. Biotechnol. 2006, 24, 905–908. (10) Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408–6418. (11) Zhang, Y. L.; Huang, Y.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2007, 129, 15448–15449. (12) Huang, Y. C.; Ge, B. X.; Sen, D.; Yu, H. Z. J. Am. Chem. Soc. 2008, 130, 8023–8029. (13) Cai, S.; Lau, C. W.; Lu, J. Z. Anal. Chem. 2010, 82, 7178–7184. (14) Cooper, V. R.; Thonhauser, T.; Puzder, A.; Schroder, E.; Lundqvist, B. I.; Langreth, D. C. J. Am. Chem. Soc. 2008, 130, 1304–1308. (15) Wetmur, J. G. Annu. Rev. Biophys. Bio. 1976, 5, 337–361. (16) Arora, A.; Nair, D. R.; Maiti, S. FEBS J. 2009, 276, 3628–3640. (17) Lang, B. E.; Schwarz, F. P. Biophys. Chem. 2007, 131, 96–104. (18) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X. F.; Sheils, C. J.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213–2222. (19) Kool, E. T. Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 1–22. (20) O’Meara, D.; Nilsson, P.; Nygren, P. A.; Uhlen, M.; Lundeberg, J. Anal. Biochem. 1998, 255, 195–203. (21) Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256–263. (22) Yu, J. H.; Ustach, C.; Kim, H. R. C. J. Biochem. Mol. Biol. 2003, 36, 49–59. (23) Green, R. J.; Usui, M. L.; Hart, C. E.; Ammons, W. F.; Narayanan, A. S. J. Periodontal Res. 1997, 32, 209–214. (24) Yuan, B. F.; Zhuang, X. Y.; Hao, Y. H.; Tan, Z. Chem. Commun. 2008, 6600–6602. 5849



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Turn-On Aptameric System for Simple and Selective Detection of

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