In Situ Hybridization Chain Reaction Amplification for Universal and

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In Situ Hybridization Chain Reaction Amplification for Universal and Highly Sensitive Electrochemiluminescent Detection of DNA Ying Chen, Jin Xu, Jiao Su, Yun Xiang,* Ruo Yuan,* and Yaqin Chai Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

ABSTRACT: In this work, we describe a new universal and highly sensitive strategy for electrochemiluminescent (ECL) detection of sequence specific DNA at the femtomolar level via in situ hybridization chain reaction (HCR) signal amplification. The DNA capture probes are self-assembled on a gold electrode. The presence of the target DNA and two hairpin helper DNAs leads to the formation of extended dsDNA polymers through HCR on the electrode surface. The in situ, HCR-generated dsDNA polymers cause the intercalation of numerous ECL indicators (Ru(phen)32+) into the dsDNA grooves, resulting in significantly amplified ECL signal output. The proposed strategy combines the amplification power of the DNA HCR and the inherent high sensitivity of the ECL technique and enables low femtomolar detection of sequence specific DNA. The developed strategy also shows high selectivity against single-base mismatch sequences, which makes our new universal and highly sensitive HCR-based method a useful addition to the amplified DNA detection arena.

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(target) and leads to the polymerization of oligonucleotides into a long nicked dsDNA molecule. Since it is an initiatortriggered reaction, it produces a comparable reduced pseudopositive result, which means that a low background can be obtained. Moreover, each copy of the initiators can trigger a HCR event, resulting in the linkage of many oligonucleotides, which shows great potential in signal amplification for DNA detection. Besides, the HCR can be operated at mild conditions. These advantages make HCR a fascinating strategy in DNA sensing applications. Very recently, Huang and coworkers30 described an optical DNA detection system which combined the amplification capability of HCR with the emission-switching property of pyrene and showed promising results. However, the complicated hairpin probe modification process with pyrene limited its wide application. In this study, based on HCR signal amplification, we propose a highly sensitive and universal strategy for electrochemiluminescent (ECL) detection of DNA sequence specific to Escherichia coli uropathogen. The presence of the target DNA sequence leads to the formation of dsDNA polymers on the sensing surface through in situ HCR. Numerous ECL indicators, Ru(phen)32+, thus can be efficiently intercalated into the grooves of the dsDNA polymers31−36 to achieve universal detection of DNA. Moreover, the ECL technique has

he development of sensitive and selective methods for the detection of trace amount of sequence specific DNA is of great importance in clinical diagnosis, food analysis, bioterrorism and environmental monitoring. Over years of development, polymerase chain reaction (PCR) has evolved as the most widely used technique for sensitive detection of DNA. With PCR, trace amount of target DNA can be amplified to detectable level.1 Although sensitive, the PCR-based technique for DNA detection encounters the problems of complicated procedures, easy contamination and high cost.2−4 In recent years, a number of methods involving the use of enzymes,5−8 indicators,9−12 and nanomaterials13−16 as signal amplification labels in connection to optical,17−22 gravimetric23,24 and electrochemical (EC)25−28 techniques as signal transduction means have been proposed as alternatives to PCR to achieve sensitive detection of DNA. Despite the attractive sensitivity of these methods, extra probe modification or conjugation steps are commonly required in these DNA detection schemes. The design of sensitive, simple and universal strategies for DNA detection will thus facilitate the construction of robust DNA biosensors. DNA nanoassembly is a simple and effective approach for signal enhancement via target−probe hybridization. If properly designed, DNAs can assemble into different structures in two or three dimensions. In 2004, Dirks and Pierce29 first introduced the concept of hybridization chain reaction (HCR) for DNA detection with PCR-like sensitivity. HCR is an enzyme-free process where a hybridization event is triggered by an initiator © 2012 American Chemical Society

Received: May 7, 2012 Accepted: August 27, 2012 Published: August 27, 2012 7750

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incubated overnight at room temperature in humidity. Then the electrode surface was rinsed with deionized water and blocked with 1 mM MCH for 2 h. After washing with PBS, the modified electrode was soaked in 10 μL target DNA solution at various concentration for 1 h. Next, the prepared electrodes were rinsed with WB, dried with N2, and incubated with a mixture of 5 μL hairpin probe H1 (1 μM) and 5 μL H2 (1 μM) in the reaction buffer for 2 h. After that, a droplet of 10 μL 2 mM Ru(phen)32+ was placed on the modified electrode and incubated for 7 h. Finally, the ECL intensity of the resulting functionalized electrode was recorded in phosphate buffer solution (0.1 M, pH 7.5) containing 20 mM TPrA from 0 to 1.2 V (versus Ag/AgCl) at the scan rate of 50 mV/s. The voltage of the photomultiplier tube (PMT) was set at 600 V in the process of detection. EC Characterizations and ECL Measurements. Cyclic voltammograms were recorded on a CHI 852C electrochemistry workstation (CH Instruments Inc., Shanghai, China). A conventional three-electrode configuration was used, with a modified gold working electrode (3 mm in diameter, CH Instruments Inc., Shanghai, China), an Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. The ECL emission was monitored by a MPI-A electrochemiluminescence analyzer (Xi’an Remax Electronic Science & Technology Co. Ltd., Xi’an, China) in phosphate buffer solution (0.1 M, pH 7.4) containing 20 mM TPrA with the voltage of the photomultiplier tube (PMT) at 600 V in detection. Gel electrophoresis was carried out on the DYCP31E electrophoretic apparatus (Beijing WoDeLife Sciences Instrument Co., Ltd.).

been demonstrated to be a simple and highly sensitive signal transduction means for the detection of different biomolecules.31,32,37,38 The integration of the HCR signal amplification with the high sensitivity of the ECL technique has been shown herein to lead to low femtomolar detection of sequence specific DNA.

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EXPERIMENTAL SECTION Chemicals and Materials. Dichlorotris(1,10-phenanthroline) ruthenium hydrate (Ru(phen)3Cl2·H2O), 6-mercapto-1hexanol (MCH) and Tripropylamine (TPrA) were purchased from Sigma-Aldrich (St. Louis, MO). Ethylenediaminetetraacetic acid (EDTA), sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride, potassium chloride, potassium phosphate dibasic, potassium phosphate monobasic were obtained from Kelong Chemical Inc. (Chengdu, China). The following buffer solutions were prepared in our laboratory. A 10 mM phosphate buffer solution (pH 7.4) was used as the DNA immobilization buffer (IB) and washing buffer (WB). Reaction buffer was 50 mM sodium phosphate buffer containing 0.5 M NaCl (pH 6.8). 1× TAE buffer was made of 40 mM Tris-acetate and 1 mM EDTA (pH 8.0). All synthetic oligonucleotides were ordered from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and the sequences were listed below. The target DNA sequence is a copy of partial region of the E. coli 16S rRNA gene (position 432−461 according to the 5′3′nucleotide sequence) specific to urinary tract infections:39 E. coli specific target DNA, 5′-TCAGCGGGGAGGAAGGGAGTAAAGTTAATA-3′; single-base mismatch DNA (sDNA), 5′TCAGCGGGGAGGAAGGGAGTAAAATTAATA-3′; NoncDNA (nDNA), 5′-GTGATCATACTTGGCAACTCGGTACCGCGC-3′; thiolated capture probe (SH−CP), 5′-SHTAT TAACTTTACTCC-3′; hairpin probe H 1 , 5′CTTCCTCCCCGCTGACAAAGTTCAGCGGGG-3′; hairpin pro be FAM-H 1 , 5′-FAM-CTTCCTCCCCGCTGACAAAGTTCAGCGGGG-3′; hairpin probe H 2 , 3′GTTTCAAGTCGCCCCGAAGGAGGGGCGACT-5′. All reagents were analytical grade and solutions were prepared using ultrapure water (specific resistance of 18 MΩ-cm). Gel Electrophoresis. All the hairpin oligonucleotides were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h before use. Then different concentration of target DNA was incubated with 1 μM FAM-H1 and 1 μM H2 in reaction buffer for 12 h. The 1% agarose gels were prepared by using 1 × TAE buffer. The gel was run at 100 V for 30 min, visualized under UV light and finally photographed with a digital camera. Pretreatment of Gold Electrode. First of all, gold electrodes were immersed in a fresh warm piranha solution (volume(concentrated sulfuric acid)/volume(30% peroxide solution) = 3:1) for 30 min. After they were rinsed thoroughly with deionized water, the electrodes were polished with 0.3 and 0.05 μm aluminum slurry and sonicated sequentially in distilled water, ethanol and distilled water for 5 min each. Then the electrodes were electrochemically cleaned in 0.5 M H2SO4 with potential scanning from 0.2 and 1.6 V until a remarkable voltammetric peak was obtained, followed by sonication again and drying with nitrogen. Fabrication of Sensors. Prior to use, all the hairpin oligonucleotides were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h. A droplet of 10 μL SH−CP (2 μM) was cast onto the pretreated electrode and

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RESULTS AND DISCUSSION

The goal of this work is to develop a new universal and highly sensitive strategy for ECL detection of sequence specific DNA via in situ HCR signal amplification. The principle is based on the formation of dsDNA polymers through in situ HCR with the presence of the target sequence and two hairpin helper DNAs, which results in the intercalation of numerous ECL probe, Ru(phen)32+, into the dsDNA polymer and generates significantly amplified ECL signals for DNA detection. As illustrated in Scheme 1, the proposed HCR-amplified protocol for DNA monitoring involves the self-assembly of the SH−CP on the pretreated gold electrode via the formation of Au−S bonds, surface blocking with MCH, in situ HCR formation of the dsDNA polymers upon addition of the target DNA and the hairpin helper probes, intercalation of Ru(phen)32+ into the Scheme 1. Illustration of the Universal and Highly Sensitive HCR-Based Strategy for ECL Detection of DNA

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weight of the product is inversely related to the amount of target, which may be due to the fact that the growth of the dsDNA polymer triggered by the target DNA will not stop until the monomers are depleted given the fixed monomer hairpins supply and enough reaction time Thus the average molecular weight of the resulting polymers is related to the amount of hairpins distributed to each initiator (target). In other words, less amount of target can form a longer DNA polymer with high molecular weight. The fabrication process of the ECL DNA biosensor was characterized by cyclic voltammetry (CV) and the resulting voltammograms were displayed in Figure 2A. The redox couple

DNA polymer, and ECL measurement of the resulting modified electrode. In the absence of the target DNA, the two hairpin probes are stable at room temperature in closed conformations. The weak interaction between the ECL probe (Ru(phen)32+) and the single stranded capture probe causes a considerably low ECL background signal. When the target DNA is present, it hybridizes with the surface-immobilized capture probe to form a partial dsDNA with a 15 nucleotide sticky end, which acts as the initiator for the HCR. Therefore, a small amount of target DNA is expected to efficiently trigger the DNA polymerization and generates a long dsDNA for the successful intercalation of Ru(phen)32+, leading to a substantial increase in the corresponding ECL intensity. The ECL signal from the intercalated Ru(phen)32+ with TPrA as a coreactant is related to the quantity of the target DNA in the testing buffer and serves as the quantitative signal for the target DNA determination. Toward the construction of the HCR-based DNA sensor, the HCR between the target sequence and the designed hairpin helper probes was first examined by gel electrophoresis. One of the monomer hairpins for generating the dsDNA polymers were labeled with fluorescein (FAM) and both monomer hairpins have an additional 6 nucleotide sticky end. In the absence of the target DNA, both monomers (FAM-H1 and H2) are closed, and only the emission band of FAM-H1 (imaged under UV-light) is observed after 30 min of gel electrophoresis (shown in Figure 1, lane 2). This UV band locates at a

Figure 2. (A) Cyclic voltammograms at the (a) bare Au, (b) CP1/Au, (c) target/CP1/Au (d) H1/H2/Target/CP1/Au electrode in 0.1 M KCl containing 1 mM [Fe(CN)6]3−/4− by scanning the potential from −0.1 to 0.6 V at a scan rate of 50 mV S1−. (B) Cyclic voltammograms recorded in 0.1 M phosphate buffer solution (pH 7.5) in the absence (a, bare Au) and presence of 20 mM TPrA on different electrodes (b) bare Au, (c) CP1/Au, (d) Target/CP1/Au, (e) H1/H2/Target/CP1/ Au, (f and e) after incubated with 2 mM Ru(phen)32+. Scan rate: 50 mV s−1.

of [Fe(CN)6]3−/4−, which is sensitive to surface chemistry, was engaged here to indicate the electrochemical behaviors of the sensor at different stage. A couple of quasi-reversible, welldefined redox peaks of [Fe(CN)6]3−/4− are observed on the pretreated bare gold electrode (curve a). After the immobilization of the capture DNA probes and surface blocking with MCH, the current responses (curve b) of [Fe(CN)6]3−/4− on the modified electrode are smaller than that of the bare electrode because the negative charges on the DNA backbone and MCH repel [Fe(CN)6]3−/4− from the electrode. In addition, the redox peaks also became irreversible, suggesting successful formation of the self-assembled layer on the gold electrode. As expected, the current response decreases further (curve c, d) after binding with the target DNA, followed by subsequent HCR, due to introduction of more negative charges on the gold surface upon formation of the dsDNA polymers. To reveal the importance of TPrA oxidation on the electrodes during the electrode modification processes, CVs of different electrode surface in 0.1 M phosphate buffer (pH 7.4) containing 20 mM TPrA were investigated. As can be seen from curve b in Figure 2B, TPrA generates a slight oxidation wave at about 0.86 V on bare gold electrode, while the peak is not observed in the absence of TPrA, indicating that the peak at 0.86 V was due to TPrA oxidation.41 After self-assembly of the capture probes onto the gold electrode, a significantly improved anodic current for TPrA oxidation is observed (Figure 2B, curve c). This current increase could be ascribed to the fact that TPrA presents in the form of TPrAH+ at pH 7.5 and approaches to the anionic phosphate backbone of DNA via electrostatic interaction,42 resulting in the preconcentration of TPrA on the electrode surface and easy transfer of proton from

Figure 1. Agarose gel electrophoresis demonstration of the target DNA initiated HCR: lane 1, 1 μM FAM-H1; lane 2, mixture of 1 μM FAM-H1 and 1 μM H2; lanes 3−5, the presence of the target DNA (from left to right, 250 nM, 50 nM, 100 nM) with a mixture of 1 μM FAM-H1 and 1 μM H2. HCR time: 12 h.

comparable distant place from the notch, indicating a lighter molecular weight and the non-HCR process between the FAMH1 and H2 monomer hairpins. Once the target is introduced, it pairs with the sticky end of FAM-H1, opens its hairpin structure via the strand-displacement interaction, and exposes a new terminus of FAM-H1 to further react with H2. In this way, each target DNA can trigger the HCR between FAM-H1 and H2 to form a long dsDNA polymer. In this case, the emission bands of high-molecular weight structures can be observed (displayed in Figure 1, lane 3−5), indicating the successful growth of the double helix. One should also note that as the HCR system would finally reach the equilibrium state in terms of kinetics and thermodynamics, the strand length in the system is different and tailing phenomenon is observed (Figure 1, lane 3−5). Consistent with previous study,40 the average molecular 7752

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TPrAH+ to phosphate ions. Therefore, the DNA assembledelectrode displays a considerable improvement on TPrA oxidation. The capture of the target DNA and subsequent HCR lead to further increase in the oxidation current of TPrA (Figure 2B, curve d and e). The dsDNA polymer-modified electrode exhibits maximum current enhancement, and contributes to the ECL emission which is related to the reaction between TPrA and Ru(phen)32+. The ECL mechanism on the modified electrode is proposed as follows according to the above results and previous reports:41−45

method, a single target DNA hybridizes with only one single capture probe to form a partial dsDNA, which limits the total signal gain from the intercalated Ru(phen)32+ and thus shows poor sensitivity. In contrast, a long dsDNA polymer structure is formed in the HCR assay and enables numerous Ru(phen)32+ molecules to be intercalated into the DNA grooves with high affinity, resulting in tremendously amplified ECL signal output. Theoretically, as each target DNA molecule has the capability of triggering the HCR between H1 and H2 to form an extended dsDNA polymer, our proposed genosensor is expected to offer high sensitivity with amplification of the intercalated ECL signal indicator Ru(phen)32+. The ECL responses of the genosensor associated with different concentration of the target DNA in the testing buffers serve as the basic value for target determination. The ECL measurements were performed in the electrolyte of phosphate buffer solution (pH 7.5) containing 20 mM TPrA under a sweep potential from 0 to 1.2 V. As shown in Figure 4,

TPrAH+(DNA) → TPrA•+ + H+(DNA) + e− TPrA•+ → (C3H 7)2 N(CHC2H5•) + H+ (Ru(phen)32 + − DNA) − e− → (Ru(phen)33 + − DNA) (Ru(phen)33 + − DNA) + (C3H 7)2 N(CHC2H5•) → (Ru(phen)32 +* − DNA) + [(C3H 7)2 N = CHC2H5]+ (Ru(phen)32 + − DNA) + (C3H 7)2 N(CHC2H5•) → (Ru(phen)3+ − DNA) + products (Ru(phen)3+ − DNA) + TPrA•+ → TPrA + (Ru(phen)32 +* − DNA)

Figure 4. (A) ECL responses of the sensor to different concentration of target DNA (a) 0 fM, (b) 25 fM, (c) 50 fM, (d) 100 fM, (e) 250 fM, (f) 1 pM, (g) 2.5 pM, (h) 10 pM, (i) 100 pM. (B) The resulting calibration plot of log c vs ECL intensity (error bars = SD, n = 3). ECL measurement conditions, as in Figure 3.

(Ru(phen)32 +* − DNA) → (Ru(phen)32 + − DNA) + hυ

The superior signal amplification capability of our protocol could be realized by comparing the analytical signal output of the traditional method and the proposed HCR assay when 2.5 pM target was incubated. As shown in Figure 3, curve b

in the absence of the target DNA, a relatively small background ECL response is observed. However, upon increasing the target DNA concentration, the ECL response increases accordingly when the amount of the hairpin probes (H1, H2) distributed to each initiator is constant. The liner range for the target DNA is from 25 fM to 100 pM with an estimated detection limit of 15 fM (3σ), which is nearly 2 orders of magnitude lower than that obtained with previous reported methods for E. coli 16S rRNA gene detection39,46,47 In addition, the relative standard deviation (RSD, n = 6) of the target DNA with different electrodes from the same batch at 1 pM level was 6.5%, indicating the good reproducibility of the ECL genosensor. The reproducibility was also examined by six repetitive measurements at 1 pM with one electrode and a RSD of 5.7% was obtained. We also investigated the stability of the proposed HCR-based DNA assay under continuous CV scanning for 7 cycles (shown in Figure 5A) and a series of sharp ECL peaks with almost constant intensity could be observed, suggesting good stability of the sensor. The selectivity of the proposed biosensor was evaluated by using the complementary, single-base mismatched and noncDNA sequences as initiators, respectively. As is shown in Figure 5B, the ECL responses of the 10 pM mismatched and noncDNA are slightly higher than that of the control experiment with the absence of the target DNA. However, the presence of a lower (10-fold) concentration of target DNA (1 pM) yields a significantly higher signal compared to the excess nontarget DNA. This comparison indicates that only the perfect matched DNA triggers the HCR process efficiently. Thus the HCR system has provided the capability of

Figure 3. ECL intensity-potential curves for DNA detection with (c) and without (b) HCR in the presence of 2.5 pM target DNA. Curve a corresponds to the background signal in the absence of the target DNA. Two mM Ru(phen)32+ was used for intercalation and ECL measurements were performed in 0.1 M phosphate buffer solution (pH 7.5) containing 20 mM TPrA.

indicates the signal output of the traditional method which is constructed with only capture probe and the target molecule, while curve c corresponds to the signal output of the proposed HCR assay which is constructed with two hairpin helper oligonucleotides. In addition, curve a is the background signal when the target DNA is not present. As expected, curve c exhibits a considerable increase in ECL intensity compared with curve b, suggesting the noticeable signal amplification effect of our HCR-based protocol. This is because in the traditional 7753

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range of DNA sequences in general with careful design of the corresponding hairpin helper DNA sequences.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-23-68252277 (Y.X. and R.Y.). E-mail: yunatswu@ swu.edu.cn (Y.X.); [email protected] (R.Y.). Notes

The authors declare no competing financial interest.

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Figure 5. (A) ECL profiles of the sensor for 100 fM target DNA under continuous 7 CV cycles. (B) Selectivity investigation for target DNA against noncomplementary (nDNA) and single-base mismatch DNA (sDNA) sequences: (a) blank solution (0 pM target DNA), (b) 10 pM nDNA, (c) 10 pM sDNA, and (d) 1 pM target DNA.

ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (Nos. 21275004, 20905062 and 21075100), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Fundamental Research Funds for the Central Universities (XDJK2012A004), and research funds from Southwest University (SWUB2008078).

discriminating the complementary target DNA sequences from nontarget sequences even with only one base difference upon DNA hybridization, suggesting advantageous selectivity of our proposed protocol.

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CONCLUSIONS In summary, we have demonstrated the construction of a universal and highly sensitive ECL biosensor for detecting sequence specific DNA at the low femtomolar level based on HCR amplification. The signal amplification relies on the HCRinduced formation of dsDNA polymers on the sensing surface and the intercalation of massive ECL indicators into the dsDNA grooves. The developed method achieves comparable or even better sensitivity against some other common amplified DNA detection schemes listed in Table 1. From this table, we Table 1. Comparison of the Proposed Assay with Some Reported Genosensors ref

detection method

DL

signal amplification strategy HCR amplification and pyreneexcimer Ru(bpy)32+-doped slica nanopaticle Glucose oxidase biocatalyzed H2O2 and luminol PCR amplification and guanine oxidation signal PCR and gold nanocomposite rolling circle amplification silver enhancement Ru(phen)32+ intercalated into HCR product

30

fluorescence

0.25 pM

48

ECL

0.1 pM

49

ECL

1 pM

50

DPV

0.81 nM

51 52 53 this work

amperometry chemiluminescence ASV ECL

60 pM 71 aM 100 aM 15 fM

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can see that the present work for DNA detection based on an enzyme-free HCR amplification technique shows about 103− 104-fold improvement on detection limit over other PCRcoupled electrochemical approach.50,51 Our method, which is free of any conjugation or labeling process, also exhibits comparable (6−60 fold-improvement) detection limit against other DNA detection schemes involving the use of fluorescent,30 nanomaterial48 or enzyme49 labels. The detection limit can be pushed down further by incorporating labels, such as enzymes or Ru(bpy)32+-doped nanomaterials into the HCR process. In addition, the HCR-based DNA detection approach is also coupled with excellent selectivity, which can be evidenced by its single base-mismatched DNA detection capability. Although, we only show the application of the proposed strategy for the monitoring of pathogen sequence specific DNA, this method can be expanded to detect a wide 7754

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dx.doi.org/10.1021/ac3012285 | Anal. Chem. 2012, 84, 7750−7755