Article pubs.acs.org/ac
Target-Triggered Quadratic Amplification for Label-Free and Sensitive Visual Detection of Cytokines Based on Hairpin Aptamer DNAzyme Probes Wenjiao Zhou, Xue Gong, Yun Xiang,* Ruo Yuan, and Yaqin Chai Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China ABSTRACT: The employment of DNAzyme probes for visual biodetections has received increasing interest recently due to the simple nature of this type of assay. However, achieving high sensitivity and detecting targets beyond nucleic acids remain two major challenges in DNAzyme-based visual detections. In this work, based on a new quadratic amplification strategy, we developed a sensitive and visual detection method for cytokines by using hairpin aptamer DNAzyme probes. The target cytokine, interferon γ (IFN-γ), associates with the aptamer sequences and unfolds the hairpin structure of the probes, leading to simultaneous recycling of the target IFN-γ (assisted by Bst-polymerase) and the DNA sequences (aided by λ exonuclease) to achieve quadratic amplification. This quadratic amplification results in the generation of numerous peroxidase-mimicking DNAzymes, which cause significantly intensified color change of the probe solution for highly sensitive detection of IFN-γ by the naked eye down to 50 pM. The proposed visual sensing method shows also high selectivity toward the target IFN-γ and can be performed in homogeneous solutions with using completely unmodified, synthetic aptamer DNAzyme probes. These distinct advantages of our developed assay protocol make it a potential platform for detecting various types of biomolecules with careful probe designs.
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preparation of probe-conjugated AuNPs,11 susceptibility to sensing environments with false positive signals (ionic strength, temperature, etc.),19,20 and potential difficulty to cause changes in interparticle distance of AuNPs for visual sensing of macromolecules. With regards to these challenges in AuNP-based visual detection approaches, a new class of functional nucleic acid probes, the peroxidase-mimicking DNAzymes, have been increasingly used recently. DNAzymes are SELEX (systematic evolution of ligands by exponential enrichment )-isolated DNA molecules that have the ability to catalyze a chemical reaction, and the catalytic activities of the DNAzymes can be regulated by specific cofactors.21−24 One of the typical examples is that the G-quadruplex sequences can associate with a cofactor, hemin, to form peroxidase-mimicking DNAzymes to catalyze the conversion of a colorless ABTS2− to a green-colored ABTS•− with the presence of H2O2.25 With the main advantages of unmodified functional nucleic acid probes and versatile sensing media, these types of DNAzymes have shown to be very useful for visual detection of different DNA targets.26−28 However, due to the nucleic acid nature of the DNAzyme probes, visual detection of proteins and other molecules by using the DNAzyme probes remains one of the major challenges for the wide application of this method.
isual detection, in which the presence of the target analyte can be directly observed by the naked eye based on color changes, has received increasing interest due to the extreme simplicity and low cost of this type of assay.1−3 Because both qualitative and semiquantitative assessment can be performed in real time without using any complicated and expensive instruments, visual detection is particularly important in field analysis and point-of-care diagnosis.4−7 Gold nanoparticles (AuNPs) have become one of the most widely used colorimetric nanoprobes up to date due to their high extinction coefficients and unique distance-dependent optical plasmon properties.8−10 The assembly of AuNPs (decrease in the interparticle distance) can lead to a strong overlap between the plasmon fields of the nearby particles, causing a red shift in the localized surface plasmon resonance and an easily observable change in color of the solution from red to blue. After the first demonstration of visual detection of DNA based on target DNA-induced assembly of two sets of probe DNA-conjugated AuNPs through sandwich DNA hybridizations,11 the color changes of AuNP solutions based on the assembly/disassembly of AuNPs have been successfully employed for colorimetric detection of small molecules,12,13 metal ions,14,15 DNA,16,17 and enzyme activities.18 Indeed, the AuNP-based visual detections have revolutionized the traditional molecular sensing methods that relied on fluorescent, chemiluminescent, and electrochemical techniques. Despite these achievements, current AuNP-based visual detection schemes still suffer from several issues, such as compromised sensitivity, time-consuming © 2013 American Chemical Society
Received: November 13, 2013 Accepted: December 4, 2013 Published: December 13, 2013 953
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Scheme 1. Principle of the New Quadratic Amplification Strategy for Highly Sensitive and Visual Detection of IFN-γ
ABTS 2− to a green-colored product ABTS •− , causing dramatically intensified color change of the solution for highly sensitive visual detection of IFN-γ. Our method features with three obvious advantages. First, the unmodified nucleic acids are used as visual probes, which makes our assay approach technically label-free. Second, highly sensitive visual detection of protein macromolecules is achieved with a new quadratic signal amplification approach by using the DNAzyme probes. Third, visual detection can be performed in one single homogeneous solution. These advantages make our approach a potential platform for highly sensitive visual detection of various biomarkers for early disease diagnostic applications.
Besides, despite a few attempts that have been exploited to improve the detection limits,29,30 visual detection of trace amounts of the targets with DNAzyme probes for early disease diagnostic applications represents another challenge. Here, by taking the advantages of aptamers in terms of versatile target binding capability, stability, high affinity, and specificity,31−35 we report on a new quadratic amplification strategy for highly sensitive visual detection of cytokines based on hairpin aptamer DNAzyme probes by using interferon γ (IFN-γ) as the model target. The presence of the target IFN-γ results in two recycling amplification cycles with the assistance of Bst-polymerase and λ exonuclease to generate numerous Gquadruplex/hemin DNAzymes to achieve quadratic signal amplification. These DNAzymes catalytically convert colorless 954
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EXPERIMENTAL SECTION Chemicals and Materials. Bst-DNA polymerase (Large Fragment), λ exonuclease, and NEB buffer 2 (10×) (50 mM NaCl, 10 mM Tris−HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9) were purchased from New England Biolabs, Inc. (Beijing, China). Carcinoembryonic antigen (CEA) was ordered from Biocell Co., Ltd. (Zhengzhou, China). Mouse IgG and IFN-γ were received from SinoBio Biotech Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO, U.S.A.). Hemin, [tris(hydroxymethy-l)aminomethane] (Tris), 2,2-azino-bis(3ethylbenzothiozoline-6-sulfonic acid) (ABTS), H2O2, and 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES) were purchased from Aladdin Reagents (Shanghai, China). The hemin stock solution (1 mM) was prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. All DNA with the following sequences were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China): hairpin aptamer probe (HAP), 5′-phosphate-TAATTCCCAATCCATGTGTTGTGGGTTGTGTTGGTTGGGGCATATTCACATGGATTGGGTTGGGCGGGATGGG-3′; primer, 5′-TCCATGTG-3′. All other reagents were of analytical grade and were used without further purification. Apparatus. A UV-2450 spectrophotometer (Shimadzu, Japan) was used to obtain the absorption spectra at room temperature in all experiments, and a canon EOS 550D camera was used to take all photographs. Polyacrylamide Gel Electrophoresis. The product solution was subjected to electrophoresis analysis on the 16% nondenaturing polyacrylamide gel. Electrophoresis was carried out in 1× TBE (pH 8.3) at a 100 V constant voltage for 60 min. After ethidium bromide staining, gels were illuminated under UV light and finally photographed by a digital camera. Quadratic Signal Amplification-Based Visual Detection of IFN-γ. The experiments were performed in 50 μL of solution containing 10 μL of HAP (10 μM), 10 μL of primer (10 μM), 15 U of Bst-DNA polymerase, 12 U of λ exonuclease, 4 μL of dNTPs (0.2 mM), and a series of targets at different concentrations. The mixture solutions were first incubated at 37 °C for 90 min. Next, 5 μL of hemin (5 μM) and 35 μL of HEPES buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, and 1% DMSO, pH 7.4) were added, and the mixture was incubated for 60 min at room temperature. Finally, ABTS2− and H2O2 were added to the mixture to reach final concentrations of 6 mM and 2 mM, respectively, at room temperature, and photographs of the solutions were taken after 5 min of color development.
strand of a double-stranded DNA (dsDNA) and exhibits no activity on single-stranded DNA or nonphosphorylated dsDNA. Upon the addition of the target IFN-γ, it associates with the aptamer loop region, leading to a conformational change of the HAP. This target-induced conformational change of the HAP thus results in the exposure of the primer recognition fragment and the active G-quadruplex sequence. Subsequently, the engaging primer anneals with the primer recognition fragment and proceeds with polymerization under the assistance of Bst-polymerase, which displaces the target IFN-γ and leads to the formation of a partially complementary DNA duplex. It should be noted that during this process the Gquadruplex sequence is not involved in the polymerization because the Bst-polymerase only shows the 5′ to 3′ polymerase activity. The displaced IFN-γ due to the polymerase extension of the engaging primer again binds with the intact HAP to initiate the IFN-γ recycling amplification cycle (recycling I in Scheme 1). Moreover, the polymerization-generated partially complementary DNA duplexes with 5′-phosphorylated, blunting termini can be recognized by λ exonuclease, which catalytically digests the 5′-phosphorylated strands of the DNA duplexes and releases the corresponding complementary strands and the G-quadruplex sequences. Importantly, the released complementary strands can hybridize with the HAP to form partially complementary DNA duplexes, and λ exonuclease further cleaves these duplexes to recycle the complementary strands to initiate recycling II as indicated in Scheme 1. By following the mechanism, the presence of the target IFN-γ can lead to two independent recycling processes, which results in a quadratic amplification format and the generation of numerous unlocked G-quadruplex sequences. These Gquadruplex sequences associate with hemin to form massive peroxidase-mimicking DNAzymes, which can catalyze the oxidation of the colorless ABTS2− to green-colored ABTS•− in the presence of H2O2. Due to the quadratic amplification nature of the proposed strategy, the presence of low levels of IFN-γ is expected to generate a large number of G-quadruplex/ hemin DNAzymes, which can potentially result in a significant color change of ABTS2− for visual detection of trace amounts of IFN-γ. For proof-of-concept demonstration of the new quadratic amplification strategy, the presence of 100 nM IFN-γ was examined by UV−vis absorption spectroscopy with/without the addition of Bst-polymerase and λ exonuclease. From Figure 1A we can see that the presence of IFN-γ (curve b) without any enzymes causes an increase in absorption intensity compared with that of the blank test (in the absence of IFN-γ, curve a). Such increase is basically due to the association of IFN-γ with the aptamer region, which unfolds the hairpin structure of the probe and exposes the active G-quadruplex sequence for subsequent formation of DNAzymes. The DNAzyme catalyzes the oxidation of the colorless ABTS2− to colored ABTS•−, causing an increase in absorbance intensity. The addition of the Bst-polymerase and the engaging primer can lead to the recycling and reuse of the target IFN-γ as previously mentioned (recycling I in Scheme 1), resulting in the formation of more active G-quadruplex sequences and further increase in absorbance intensity (curve c). Most importantly, dramatic increase in absorbance intensity is observed (curve d) with the addition of λ exonuclease (formation of quadratic amplification). These results indicate that the colorimetric signal output of the presence of the target IFN-γ can be significantly improved with the introduction of the quadratic amplification
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RESULTS AND DISCUSSION Our quadratic amplification IFN-γ sensing principle is illustrated in Scheme 1. In the probe design, the G-quadruplex sequence (the mandarin blue part in Scheme 1), the IFN-γ binding aptamer sequence (the red loop region), and the primer recognition fragment (the cyan part) are incorporated into a hairpin aptamer probe (HAP) in an initially locked format by hybridizing with their partially complementary sequences. In the absence of IFN-γ, the formation of DNAzymes or the initiation of the polymerization by Bstpolymerase are prohibited. Moreover, the HAP sequence is designed in such a way that it is resistant to catalytic digestion by λ exonuclease in the absence of IFN-γ, due to the fact that the λ exonuclease only selectively digests the 5′-phosphorylated 955
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Figure 2. Effect of the quadratic reaction time on the UV−vis absorption intensity of the proposed quadratic amplification method for IFN-γ detection. Reactions were performed at 37 °C.
reaction time of 90 min was selected for subsequent experiments. To evaluate the dependence of the UV−vis absorption intensity of the probe solution upon the concentration of the target, different concentrations of IFN-γ were measured with the proposed method. From Figure 3A we can see that the
Figure 1. (A) Dependence of the signal output upon the presence of different enzymes monitored by UV−vis spectroscopy: (a) HAP, Bstpolymerase (15 U), and λ exonuclease (12 U) as the blank; (b) HAP and IFN-γ (100 nM); (c) HAP, IFN-γ (100 nM), and Bst-polymerase (15 U); (d) HAP, IFN-γ (100 nM), Bst-polymerase (15 U), and λ exonuclease (12 U). (B) PAGE characterization of IFN-γ-induced quadratic recycling amplification reactions: lane 1, HAP (2 μM); lane 2, HAP (2 μM), Bst-polymerase (15 U), and λ exonuclease (12 U); lane 3, HAP (2 μM), IFN-γ (100 nM), and Bst-polymerase (15 U); lane 4, HAP (2 μM), IFN-γ (100 nM), Bst-polymerase (15 U), and λ exonuclease (12 U). The mixtures were incubated at 37 °C for 60 min.
approach. The quadratic amplification reactions were further verified by using polyacrylamide gel electrophoresis (PAGE), and the results were in agreement with the proposed mechanism (Figure 1B). In the absence of IFN-γ, the hairpin structure of HAP is maintained even with the addition of Bstpolymerase and λ exonuclease, and the UV bands locate at a comparable distance from the notch (lanes 1 and 2), indicating a lighter molecular weight and nonpolymerization reaction initiated by Bst-polymerase. However, once IFN-γ is introduced, it binds with the aptamer region of HAP, leading to the unfolding of the hairpin structure and exposure of the primer recognition region. In this case, the primer hybridizes with the primer recognition region and the polymerization is initiated by Bst-polymerase and partially complementary DNA duplexes are produced accordingly. These DNA duplexes with high molecular weight exhibit a clear band above that of HAP (lane 3). With the addition of the λ exonuclease, the DNA duplexes are digested to release the G-quadruplex sequences and to cause cyclic cleavage of HAP. Consistently, a new, low molecular weight band corresponding to the G-quadruplex sequences and the disappearance of the HAP band due to cyclic enzyme cleavage are observed (lane 4). These PAGE results indicate the successful proceeding of the quadratic amplification reactions. One of the key factors that affects the assay performance of the proposed method is the quadratic reaction time. In order to achieve optimal assay conditions, the quadratic reaction time was optimized. For this purpose, the effect of the reaction time on the signal output of the proposed method was investigated by monitoring the UV−vis absorption intensity of the probe solution with the presence of IFN-γ (100 nM), Bst-polymerase (15 U), and λ exonuclease (12 U) at a time interval of 15 min from 15 to 120 min. As displayed in Figure 2, the absorption intensity of the mixture increases rapidly with increasing reaction time in the range from 15 to 90 min and reaches a plateau thereafter. To ensure complete quadratic reactions, the
Figure 3. (A) Typical UV−vis absorption spectra for quadratic signal amplification-based IFN-γ detection at various concentrations: 0, 0.005, 0.05, 1, 10, 25, 50, 75, and 100 nM (from bottom to top). Inset: the corresponding calibration plot of the concentration of the target IFN-γ vs the absorption intensity. Error bars: SD, n = 3. (B) Photograph for visual IFN-γ detection at different concentrations: (a) 0, (b) 0.05, (c) 1, (d) 25, (e) 75, and (f) 100 nM. The amount of Bstpolymerase, 15 U; λ exonuclease, 12 U; quadratic reaction time, 90 min.
UV−vis absorption intensity increases accordingly with increasing concentration of IFN-γ from 0 to 100 nM. The inset of Figure 3A shows the corresponding calibration plot of the concentration of IFN-γ versus the UV−vis absorption intensity, in which the detection limit was calculated to be 1.5 pM according to the responses of the blank tests (n = 11) plus 3 times the standard deviation (3σ). The color change of the presence of various concentrations of IFN-γ was also monitored by the naked eye to achieve visual detection. As illustrated in Figure 3B, the green color of the solution was gradually intensified with elevated concentration of IFN-γ from 0 to 100 nM, which is in agreement with the UV−vis measurements. Importantly, the presence of as low as 50 pM IFN-γ can be easily identified with the naked eye according to the distinct color difference between the sample and the blank test (Figure 3B, b vs a). Such low visual detection limit (50 pM) for IFN-γ 956
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is comparable to those methods based on electrochemical36−38 or optical transduction means,39,40 due to the synergistic effect of the quadratic amplification of the colored signal output. Besides the impressive visual detection limit, our method is essentially simple by avoiding the involvement of any probe immobilization or conjugation step and any complicated signal transduction instruments, which are common requirements in electrochemical or optical biodetections. The selectivity of the proposed method is examined by monitoring the color change of the solution with the presence of IFN-γ against other control molecules, BSA, IgG, and CEA. As shown in Figure 4, the presence of the control molecules
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-23-68252277. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by NSFC (Nos. 21275004, 20905062, 21075100, and 21275119) and the New Century Excellent Talent Program of MOE (NCET-12-0932).
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Figure 4. Selectivity investigation of the proposed visual detection method for IFN-γ against BSA, IgG, and CEA all at identical concentration of 50 pM.
(50 pM) causes insignificant color changes of the solutions while the addition of the target IFN-γ (50 pM) results in a clear green color change of the probe solution, revealing that the quadratic amplification approach is highly selective. Such high selectivity can be related to the highly specific binding capability of the aptamer sequence to IFN-γ. In other words, only the presence of IFN-γ can associate with the aptamer region and unfold the hairpin structure of HAP to initiate the quadratic amplification cycles to achieve highly sensitive visual detection of IFN-γ.
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CONCLUSIONS In conclusion, we have developed a quadratic amplification strategy by integrating two independent recycling cycles for highly sensitive visual detection of IFN-γ. Due to the significant quadratic signal amplifications, the presence of IFN-γ leads to the generation of numerous G-quadruplex/hemin DNAzymes, which result in intensified color change of the probe solution for visual detection of IFN-γ down to 50 pM with the naked eye. The developed method also exhibits high selectivity toward IFN-γ against other interfering molecules. Besides, our assay protocol requires only one step to realize quadratic amplification for sensitive visual detection of IFN-γ without the need for multiple conjugation steps required in common signal amplification assays. Compared with other traditional antibody-based sandwich immunoassays or aptamer-based assays for IFN-γ, the proposed approach has the advantages of high sensitivity, rapid signal readout, and simplicity. Moreover, this quadratic signal amplification approach can be potentially extended for visual detection of other proteins at low levels with proper probe designs, which enables the developed method to be a universal aptamer-based visual sensing platform for a variety of target analytes. 957
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