Cooperative Amplification-Based Electrochemical Sensor for the

Aug 2, 2013 - (1-3) Of the current methods for DNA detection, electrochemical DNA ... respective 0-, 8-, 10-, and 12-mer nucleotides complementary to ...
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Cooperative Amplification-Based Electrochemical Sensor for the Zeptomole Detection of Nucleic Acids Liping Qiu, Li Qiu, Zai-Sheng Wu,* Guoli Shen, and Ru-Qin Yu* State Key Laboratory of Chemo/Biosensing and Chemometrics, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, People’s Republic of China S Supporting Information *

ABSTRACT: In this work, we developed a multiple-amplificationbased electrochemical sensor for ultrasensitive detection of nucleic acids using a disease-related sequence of the p53 gene as the model target. A capture probe (CP) with a hairpin structure is immobilized on the electrode surface via thiol−gold bonding, while its stem is designed to contain a restriction site for EcoRI. In the absence of target DNA, the probe keeps a closed conformation and forms a cleavable region. After treatment with EcoRI, the target binding portion (loop) plus the biotin tag can be peeled off, suppressing the background current. In contrast, the CP is opened by the target hybridization, deforming the restriction site and forcing the biotin tag away from the electrode. On the basis of the biotin−streptavidin complexation, gold nanoparticles (GNPs) modified with a large number of ferrocene-signaling probes (Fc-SPs) are captured by the resulting interface, producing an amplified electrochemical signal due to the GNP-based enrichment of redox-active moieties. Furthermore, Fc tags can be dragged in close proximity to the electrode surface via hybridization between the signaling probes and the CP residues after EcoRI treatment, facilitating interfacial electron transfer and further enhancing the signal. With combination of these factors, the present system is demonstrated to achieve an ultrahigh sensitivity of zeptomole level and a wide dynamic response range of over 7 orders of magnitude. tion of the “signal-on” model and enzyme-amplified signal effect, the E-DNA sensor is able to detect a considerably low amount of target.11 Liposomes labeled with horseradish peroxidase (HRP) have been used to amplify DNA sensing events, achieving impressive signal amplification.12 Nevertheless, it is challenging to suppress the background, and few elegant techniques have been reported to facilitate the signalrelated electron transfer, especially when the target DNA is at ultralow concentration. Compared with bioactive enzymes, gold nanoparticles (GNPs) have many attractive merits, including high stability, low cost, good biocompatibility, and flexible modification.13−16 Herein, we report a signal-on E-DNA senor for the highly sensitive detection of DNA targets based on the ingenious combination of GNP-dependent enrichment of redox-active moieties, electron-transfer-determined signal amplification, and background suppression. A specific p53 gene sequence, whose mutation is closely related to cancer, was chosen as the analyte model.17−19 To achieve this goal, streptavidin-conjugated gold nanoparticles capped with multiple ferrocene (Fc)-signaling probes (Fc-GNP-SA; the fabrication details are described in the Experimental Section) are used as reporters to signal and

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he ultrasensitive detection of DNA sequences provides valuable information for genetic disease diagnosis, forensic identification, and food safety monitoring.1−3 Of the current methods for DNA detection, electrochemical DNA sensors may be the most promising, due to their intrinsic advantages of high sensitivity, low cost, rapid response, and easy miniaturization.4 To develop a sensitive electrochemical DNA sensor, an important consideration is the choice of an effective strategy to signal the target events. One of the commonly used methods relies on the conformational change of the recognition probe induced by the target hybridization.5−9 The E-DNA sensor is such an example.5,7−9 The E-DNA sensor, as an electrochemical analogue of the fluorescent “molecule beacon”,10 usually consists of a surface-confined hairpin probe labeled with an electroactive reporter. On hybridization with target DNA, the conformation of the probe changes from the stem−loop structure to a rigid linear duplex, extending the electroactive reporter away from the electrode surface and resulting in a current signal change which can be used to quantify the target. However, such an E-DNA sensor often suffers from a limitation of inadequate sensitivity, mainly due to the relatively small conformational variation and the undesirable “signal-off” response mode.8 To improve the sensitivity of the E-DNA sensor, research can be directed toward three aspects: amplification of the target hybridization event, improvement of the electron transfer efficiency, and suppression of the background. With combina© 2013 American Chemical Society

Received: April 30, 2013 Accepted: August 2, 2013 Published: August 2, 2013 8225

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Table 1. Oligonucleotides Designed in the Present Studya

CP has a −SH moiety at the 3′-terminus for subsequent self-assembly on the gold electrode surface and a biotin moiety at the 5′-terminus for specific complexation with streptavidin. TON has a −SH moiety at the 5′-terminus for functionalization of the gold nanoparticle. SP1, SP2, SP3, or SP4 has an −NH2 moiety at the 5′-terminus for modification of Fc. The point mutation of M is indicated in bold type. NH2-CP has the same sequence as CP, but with a 5′-end −NH2 moiety for Fc modification. a

EcoRI (15 U/μL) was obtained from Takara Biotechnology. Ferrocenemonocarboxylic acid and N-hydroxysuccinimide (NHS) were purchased from Acros Organics, while N-[3(dimethylamino)propyl]-N′-ethylcarbodiimide (EDC) was received from Sigma. All other chemical reagents involved were of analytical grade. Deionized and sterilized water (resistance >18 MΩ·cm) was used throughout. Preparation and Characterization of Fc-GNP-SA Conjugates. The solution of 13 nm GNPs was prepared following the previous method,15 and its concentration, which was calculated via the Lambert−Beer law with an extinction coefficient of 2.7 × 108 M−1·cm−1,21 was about 2.9 nM. The asprepared GNPs were functionalized with TON, according to the protocol described previously with slight modification.14 In short, the GNP solution was concentrated 2-fold by centrifugation. Then 100 μL of 10 μM TON was injected into 1 mL of the concentrated GNP solution and incubated with gentle stirring for 24 h at room temperature. Subsequently, 130 μL of 3 M NaCl solution was added slowly and allowed to incubate for at least 24 h. After that, excess TONs were removed by centrifugation two times. The precipitate was redispersed in 1 mL of phosphate-buffered saline (PBS; pH 7.4) and then mixed with 100 μL of 70 nM streptavidin (diluted with PBS (pH 7.4)) for 1 h on a vortex stirrer. The streptavidin-to-nanoparticle ratio was about 1:1, given that the loss of gold nanoparticles during the centrifugation could be neglected. Finally, the resulting conjugates were mixed with an equal volume of Fc-singaling probes (Fc-SPs; 1000 nM), which were synthesized via coupling of the succinimide ester of ferrocenecarboxylic acid with the 5′-amine of the signaling probes.22 After incubation for 1 h and removal of the excess FcSPs, the final Fc-GNP-SA complexes were obtained. The loading capacity of TONs on each GNP was measured with a fluorescence method.23 First, TONs labeled with 3′-end TAMRA (carboxytetramethylrhodamine) fluorophore (TMRTONs) were immobilized on the GNP surface according to the above-mentioned procedure. After removal of unbound TMRTONs, mercaptohexanol (MCH; final concentration of 20 mM) was added. The mixture was incubated at room

amplify the target DNA hybridization event. A restriction site of endonuclease enzyme (EcoRI)20 is introduced in the stem region of the capture probe to guarantee a low background, and an Fc-dragging strategy through DNA hybridization between the signaling probe and the capture probe is used to promote the interfacial electron transfer. With cooperative integration of multiple elements (signal-on architecture, restriction enzymatic cleavage, gold nanoparticles, and dragging mechanism) to increase the signal-to-noise ratio, the sensor achieves an ultrahigh sensitivity (a detection limit of 5 zmol) and a wide linear response range (from 1 nM to 1 fM). In this paper we describe the working principle, optimization of factors influencing the current signal, and the analytical performance. As a proof-of-concept, the proposed biosensing scheme is expected to provide a powerful platform for DNA analysis in research and medical diagnosis.

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EXPERIMENTAL SECTION Chemicals. Oligonucleotides designed in this study were synthesized by Invitrogen Biotechnology, and their sequences are listed in Table 1. The loop portion (lowercase) of the capture probe (CP) is completely complementary to the target DNA (T), while the double-stranded stem (shaded) contains a restriction site for EcoRI. Signaling probes 1, 2, 3, and 4 (SP1, SP2, SP3, and SP4) all have two segments: one (in gray) is complementary to the thiolated oligonucleotide (TON); the other (underlined) possesses respective 0-, 8-, 10-, and 12-mer nucleotides complementary to the 3′-end region of CP (boldface). SP2 was used throughout unless otherwise specified. All the oligonucleotide products (freeze-dried powder) were first dissolved with TE buffer (10 mM Tris− HCl (pH 8.0) containing 1 mM EDTA). Then they were diluted to a certain concentration with working buffer (50 mM Tris−HCl (pH 7.4) containing 10 mM MgCl2 and 100 mM NaCl). In consideration of the possible disturbance of 1,4dithiothreitol (DTT) to the thiol−gold self-assembled monolayer,14 the working buffer used for EcoRI cleavage reaction in the present study was somewhat different from the standard recipe by omitting DTT. 8226

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Scheme 1. (A) Design of the Capture Probe Which Can Recognize the Target DNAa and (B) Schematic Illustration of This Sensor

a

The recognition site for EcoRI is illustrated in the box, while the cleavage position is indicated by the dashed line.

incubation in a water-saturated atmosphere overnight, the resulting electrode was washed with ultrapure water. Finally, the electrode surface was passivated by MCH (10 mM) for 10 min. Thus, a self-assembled monolayer on the electrode surface for DNA detection was obtained. Detection of the Target DNA. The target sample (10 μL) at a specific concentration was pipetted onto the self-assembled electrode surface and allowed to react at 37 °C for 60 min. Then the electrode was rinsed in working buffer with gentle stirring for 10 min, followed by incubation with EcoRI (0.75 U/ μL) at 37 °C for 120 min. After being washed with 100 mM PBS, the resulting electrode was covered with 20 μL of FcGNP-SA at room temperature for 10 min. Finally, the electrode was washed with 100 mM PBS (pH 7.4) and kept in this buffer for at least 20 min to facilitate DNA hybridization. Electrochemical measurements were performed on a CHI 760 B electrochemical workstation (Shanghai,China) at room temperature using a normal three-electrode system consisting of the gold working electrode, a platinum counter electrode, and a KCl saturated calomel reference electrode (SCE). The detection buffer was 20 mM PBS (pH 7.4) containing 0.1 M KClO4 (a weak nucleophile, used as the electrolyte to avoid the

temperature with gentle shaking for 12 h and then centrifuged to get rid of the GNP aggregates. The fluorescence intensity of displaced TMR-TONs in the supernatant was recorded and converted to the concentration of the TMR-TONs by using a standard linear calibration curve. Finally, the average number of TONs per particle was 71 ± 6, which was calculated by dividing the measured TON concentration by the original GNP concentration. The number of signaling probes captured on each GNP via hybridization with TONs was 22 ± 1, which was measured with the same method, except that nonfluorescent TONs and fluorescent SP2, whose NH2 moiety at the 5′-end was replaced with a TAMRA fluorophore, were used. Fabrication of the Sensing Interface. The gold electrode (99.99% polycrystalline, ∼2 mm diameter, CH Instrument Inc.) was treated according to the following procedure: First, the electrode was polished with 0.05 μm γ-alumina suspension on a microcloth for 5 min, and then it was successively sonicated in ultrapure water, ethanol, and ultrapure water for 2 min each. Second, the electrode was soaked in fresh piranha solution (3:1 H2SO4/H2O2) for 20 min, followed by sonication in ultrapure water for 2 min. Third, the pretreated gold electrode was inverted and covered with 15 μL of 1 μM CP solution. After 8227

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Figure 1. Cyclic voltammograms (A) and differential pulse voltammograms (B) of the present system with (curve a) and without (curve b) 1 nM target DNA.

instability of the ferrocenium).24 Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and ac impedance measurements were employed to validate the present sensing system. The reported DPV curves were background-subtracted with Sigma Plot 10.0 via extrapolation to the baseline.25

extent. This signaling strategy is expected to result in a powerful sensor for the ultrasensitive detection of DNA hybridization. The successful fabrication of this sensing interface has been characterized with ac impedance analysis, and the experimental results are shown in Figure S1 (Supporting Information). The relationship between CV response and the scan rate was investigated. The data demonstrate the typical feature of the surface-bound electrochemical process, confirming that Fc is confined to the electrode surface after biotin−streptavidin complexation (Figure S2, Supporting Information). The surface density of the immobilized CP was calculated from the amount of the redox charge (Q) produced by the Fc-modified CP (whose 5′-end was labeled with an NH2 moiety for Fc modification) within the CV process using a previously reported fomula27 with adaptive adjustment: TCP = Q/nFA (n is the number of electrons per mole for reduction, F is the Faraday constant, and A (62.5 × 10−3 cm2) is the electrode surface area20). Q obtained from the ampere-hour (A h) of the CV measurement is ∼5.1 × 10−8 C. Given that all Fc tags captured on the electrode surface were electrochemically active, the surface density of immobilized CP is about 8.5 × 10−12 mol/cm2 (corresponding to 5.1 × 1012 molecules/cm2), which is compatible with that of the previous reports.27−29 To confirm the feasibility of the present sensing system, cyclic voltammograms and differential pulse voltammograms were collected to compare the current signal induced by the target with the background signal under identical conditions. As shown in Figure 1A, upon addition of the target DNA, a couple of well-defined redox peaks are obtained in CV curves at 0.16 and 0.21 V (vs SCE, curve a), exactly corresponding to the typical redox potential range of Fc. In contrast, no detectable current peaks appear for the blank. Similar results but better differentiation can be obtained with DPV. As shown in Figure 1B, a remarkable DPV peak at 0.18 V (vs SCE, curve a) is caused by the target, while the background signal (curve b) can be negligible. The calculated signal-to-noise ratio of ∼30 is about 3 times and 6 times larger than those of the works reported by Fan11 and Zhang,26 respectively. The measured data indicate that the present scheme provides a promising platform for DNA detection. Optimization of Signaling Probes. The approach of the Fc tag to the electrode surface is dependent on the hybridization between Fc-SP and the CP residue left on the electrode after enzymatic cleavage. To verify the benefit of this dragging strategy and to improve its performance, four

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RESULTS AND DISCUSSION Working Principle. The working principle is illustrated in Scheme 1. A hairpin DNA sequence, whose loop is complementary to the target molecule, serves as the CP. Its stem contains a restriction site for EcoRI. CP is labeled with a thiol moiety at the 3′-end for self-assembly onto the gold electrode surface and with a biotin moiety at the 5′-end to introduce signaling reporters. In the absence of the target, CP folds into a stem−loop structure, forming a cleavable restriction site for EcoRI. After treatment with EcoRI, the target binding segment of CP plus the biotin tag is cleared away, preventing the subsequent signaling reactions from occurring and thus resulting in a reliably suppressed background current (the CP residue left on the electrode after the washing process is designed to further amplify the electrochemical signal, vide infra). In contrast, upon target hybridization, the CP unfolds, deforming the EcoRI restriction site; hence, EcoRI is unable to cleave the CP sequence. Since the CP/target DNA hybridization forms a rigid duplex, the biotin moiety is forced away from the electrode surface and becomes accessible to the FcGNP-SA. With the large number of Fc-SPs attached to each nanoparticle captured on the electrode by the target hybridization-induced biotin−streptavidin interaction, the electrochemical current signal can be significantly amplified. However, due to the electrostatic repulsion between the signaling probes and the CP-modified electrode interface, the Fc tags may fail to contact the electrode surface efficiently, thus limiting the enhancement of the electrochemical signal. To meet this challenge, the Fc tag is dragged close to the electrode through hybridization of the 5′-end segment of Fc-SP (underlined, Table 1) to the 3′-end segment of CP (boldface, Table 1), which is left on the electrode surface only if enzymatic cleavage occurs. In this case, Fc tags are drawn in close proximity to the electrode surface, facilitating interfacial electron transfer. Here, the CP residue functions as a fixer to tie Fc-SP, eliminating the need for introduction of an additional probe.26 This can make additional room for the functional probe (CP) to occupy the electrode surface, improving its analysis capability to some 8228

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number of SP2s owing to the enrichment effect of GNPs, but also a certain number of the terminal Fc tags can approach the electrode surface to promote the interfacial electron transfer. Because of its high target-to-blank ratio (27.9), SP2 was used as the optimal signaling probe in the present work. Detecting the Target DNA. To assess the analysis performance of the present sensing system, a series of target samples at different concentrations were prepared and DPV was conducted to record the response currents. As shown in Figure 3, the peak currents increase dynamically with increasing

signaling probes (termed SP1, SP2, SP3, and SP4) containing different numbers of nucleotides complementary to CP were designed. Under the present conditions, the melting temperatures (Tm), which are calculated using bioinformatics software (http://www.bioinfo.rpi.edu/applications/) for the hybrids of CP with SP1, SP2, SP3, and SP4, are 0, 16.5, 31, and 38.9 °C, respectively. In theory, CP is unable to hybridize with SP1 or SP2 at room temperature (25 °C), while its hybridization with SP3 or SP4 is achievable. The detection capabilities of this sensing system containing each of these four probes were individually evaluated in the presence and absence of the target DNA. As shown in Figure 2, the current peak intensity induced

Figure 3. DPV responses of the sensing system for the target DNA (T) at varying concentrations. Inset: linear relationship between the peak current and the logarithm of the target concentration. The error bars represent the standard deviation of three repetitive measurements.

Figure 2. Differential pulse voltammograms of the present system with different signaling probes in the presence (solid circels) and absence (open circle) of 100 pM target DNA. The error bars represent the standard deviation of three repetitive measurements.

target concentration. A good linear relationship between the peak current value (IP) and the logarithm of the target concentration (c) ranging from 1 fM to 1 nM is achieved. The peak current responding to the target at higher or lower concentration is beyond the linear response range. The linear regression equation is IP = −32.12 + 47.60 log c, with a correlation coefficient of 0.9940, and the detection limit is 0.5 fM, amounting to 5 zmol of DNA molecules in a 10 μL sample. This ultrahigh sensitivity prevails over all previously reported electrochemical DNA sensors listed in Table S1 (Supporting Information). Such an excellent analysis performance may be attributed to several factors. First, the introduction of a restriction site in the stem of the recognition probe and the high cleavage activity of EcoRI can reliably suppress the background signal, as indicated by a nearly 4-fold increase in the background current without EcoRI treatment (Figure S4, Supporting Information), mainly because of the CP/Fc-GNPSA interaction resulting from the opening of some CP hairpin structures during the washing step. Second, GNP is used as a functional carrier for SPs. On the basis of the enrichment effect of GNP (22 ± 1 Fc-SPs are attached onto each functional nanoparticle), multiple Fc moieties can be captured onto the sensing interface even if one target binding event is involved, enhancing the response signal significantly. Third, by using hybridization between SPs and CP residues, Fc tags can be dragged into close proximity of the electrode surface, promoting interfacial electron transfer and further enhancing the signal gain, as required for trace detection of the target molecules. Furthermore, the CP residue left on the electrode surface after EcoRI treatment is designed to act as a fixer for the

by the target DNA in the case of SP1 is merely 56% of that with SP2, while the background in both cases is negligible. Data for SP1 and SP2 indicate that the signal-to-noise ratio will be enhanced if the signaling probe is designed to have a region complementary to CP to “drag” the Fc tag close to the electrode, improving the response capability of this sensing system. However, further extension of the complementary region of the signaling probe to CP (e.g., SP3 and SP4) causes the redox peak to increase regardless of whether the target DNA is present, resulting in a deteriorated signal-to-noise ratio (∼3.6 and ∼1.7, respectively). It is presumed that SP3 and SP4 themselves can hybridize to the CP residue even if no biotin− streptavidin complexation occurs in the absence of the target DNA, inevitably leading to an increased background. On the other hand, SP2 itself cannot form a stable hybrid with the CP residue due to the relatively short complementary region. If endonuclease cleavage of some CPs is protected by the target DNA hybridization, the Fc tag will be dragged to the electrode surface, because the functional gold nanoparticle is tightly “captured” by the CP/target DNA duplex on the basis of the biotin−streptavidin complexation and the simultaneous multivalent interactions promote SP2s’ hybridization with the CP residues. A similar multivalent effect has also been verified by other works.22,30 It was demonstrated that, without the simultaneous multivalent effect aided by the functional carrier (streptavidin/GNP conjugate), SP2s themselves cannot hybridize with CPs even after 2 h of incubation (Figure S3, Supporting Information). In this design, not only can each target hybridization event cause the adhesion of a considerable 8229

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strategy to bring the Fc reporter close to the electrode, as well as other design features. First, a hairpin sequence is used as the capture probe with a restriction site introduced into its stem segment. With high efficiency and high fidelity of EcoRI, the enzymatic cleavage reaction only occurs on those probes that retain their stem−loop structure without capturing the target, leading to a reduced background signal. Second, the residues remaining after endonuclease treatment served as fixers to bring the Fc moieties in close proximity to the electrode surface, favoring interfacial electron transfer. Signal amplification also originated from the enrichment effect of GNP, which triggers a large number of Fc-signaling probes even if one target binding event occurs. With its high sensitivity, low cost, and good reproducibility, this electrochemical sensor is expected to provide a promising platform for biomolecule analysis and association studies.

Fc moiety, avoiding the hassle of adding exogenous adjunct probes26 and maintaining the valid occupancy of CPs on the sensing surface.27,28 As demonstrated in Figure S5 (Supporting Information), without the “dragging” strategy, the detection limit of this sensor is only 100 fM, over 2 orders of magnitudes poorer than that with the “dragging” strategy. In short, all these factors work cooperatively to accomplish the extremely high sensitivity of this sensing scheme. To estimate the reproducibility of the present system, three target samples at different concentrations (102, 103, and 104 fM) within the dose−response curve were investigated, and the data are displayed in Table S2 (Supporting Information). The maximum relative standard deviation obtained is only 10.0% for three repetitive measurements, indicating a desirable reproducibility for DNA detection. The application of this sensing system for DNA assay in the complicated environment (10% blood serum) was validated, and the result is shown Figure S6 (Supporting Information). Selectivity of the Present System. To evaluate the selectivity of this sensing system, a one-base-mismatched DNA (M) was interrogated under identical conditions. As shown in Figure 4, the current signal caused by M is no more than 63%

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-731-88821916. Fax: 86-731-88821916. E-mail: [email protected] (Z. S. Wu); [email protected] (R. Q. Yu). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21275002, 21190041, 20905022, 91117006, and 21205039) and the National Key Basic Research Program of China (Grant No. 2011CB911000).

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Figure 4. Selectivity of the present system. The DPV response of the fully complementary target DNA (T) is defined as 100%, while that of the one-mismatch DNA (M) is the relative value. The concentrations of T and M are both 100 pM. The error bars represent the standard deviation of three repetitive measurements.

of that induced by the fully complementary T. The ability to distinguish a point mutation using this sensor is comparable to that described in several reports.8,31,32 It has been reported many times that molecular beacons behave well in discriminating single-nucleotide polymorphisms (SNPs) in homogeneous systems.10 However, interfacial hairpin structures often show poor performance in this regard, probably due to the surfaceinduced instability on the stem structure. Presumably, increasing the distance between the stem−loop probe and the electrode surface by using underlayers and/or spacers would reduce the interfacial disturbance on the conformation of the probe,11 thereby improving the selectivity.

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CONCLUSIONS In conclusion, we have developed a signal-on sensor for the highly sensitive detection of the p53 gene sequence. This sensor achieves an ultralow detection limit of zeptomole and a wide dynamic range of over 7 orders of magnitude. The excellent analysis capability can be attributed to the dragging 8230

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