Amplified Detection of DNA through the Enzyme-Free Autonomous

Dec 14, 2011 - The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 × 10–13 M. The optimized sys...
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Amplified Detection of DNA through the Enzyme-Free Autonomous Assembly of Hemin/G-Quadruplex DNAzyme Nanowires Simcha Shimron, Fuan Wang, Ron Orbach, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: An enzyme-free amplified detection platform is described using the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying label. Two hairpin structures that include three-fourths and one-fourth of the HRP-mimicking DNAzyme in caged, inactive configurations are used as functional elements for the amplified detection of the target DNA. In the presence of the analyte DNA, one of the hairpins is opened, and this triggers the autonomous cross-opening of the two hairpins using the strand displacement principle. This leads to the formation of nanowires consisting of the HRP-mimicking DNAzyme. The resulting DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent readout of the sensing processes (the term “enzyme-free” refers to a protein-free catalyst). The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 × 10−13 M. The optimized system acts as a versatile sensing platform, and by coaddition of a “helper” hairpin structure any DNA sequence may be analyzed by the system. This is exemplified with the detection of the BRCA1 oncogene with a detection limit of 1 × 10−13 M.

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wires, consisting of the HRP-mimicking hemin/G-quadruplex DNAzyme using the rolling circle amplification process,37,38 was reported to yield a highly sensitive detection platform for DNA. In the present study we describe an enzyme-f ree amplification platform that involves the use of functional nucleic acid nanostructures that upon recognizing the target DNA trigger an autonomous cross-opening process that yields the formation of the hemin/G-quadruplex DNAzyme wires. The resulting nanowires enable, then, the colorimetric or chemiluminescent readout of the sensing process. We implement the method to analyze the BRCA1 oncogene. The sensitivity of this detection platform is comparable to that of the enzyme-mediated isothermal DNA “machines”.

he amplified sensitive detection of DNA attracts substantial research efforts due to the broad possible applications in medical diagnosis, detection of environmental pollutants, controlling food quality, homeland security, and forensic analyses.1−4 Different optical,5−7 electronic,8−12 and microgravimetric13,14 amplified sensing platforms for the analysis of DNA were developed. These included the application of enzymes,15,16 metal nanoparticle catalysts,17,18 and plasmonic nanoparticles19,20 as amplifying labels. Catalytic nucleic acids (DNAzymes or ribozymes) find growing interest as amplifying labels for DNA sensing events.21 The flexibility to encode in the base sequences of DNA functional and structural information, together with the reduced nonspecific binding properties of nucleic acids, provide unique features for bioanalytical applications. For example, metal-dependent DNAzymes were used for the amplified detection of DNA.22−26 Specifically, the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme27 was extensively used as a catalytic label for the amplified colorimetric28−30 or chemiluminescence31−33 detection of DNA. Also, the hemin/ G-quadruplex was used as an electrocatalytic34 or optical35 label for the electrochemical or surface plasmon resonance (SPR) detection of DNA. DNA “machines” that activate the autonomous isothermal replication of DNAzyme units have been suggested as possible routes to amplify DNA detection and to provide alternative routes to the polymerase chain reaction (PCR) process. For example, the autonomous synthesis of the hemin/G-quadruplex DNAzyme on a nucleic acid track, as a result of recognizing the target DNA, and using the polymerase-dNTP/nicking enzyme complex as a catalyst, was reported to yield the ultrasensitive detection of DNA.36 Similarly, the polymerase-stimulated synthesis of DNA nano© 2011 American Chemical Society

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EXPERIMENTAL SECTION Materials. Hemin was purchased from Porphyrin Products (Logan, UT) and used without further purification. A hemin stock solution was prepared in DMSO and stored in the dark at −20 °C. Fetal bovine serum was purchased from Biological Industries, Kibbutz Beit Haemek. All other chemicals were obtained from Sigma-Aldrich and used as supplied. Ultrapure water from a NANOpure Diamond (Barnstead) source was used in all of the experiments. All oligonucleotide sequences were purchased from Integrated DNA Technologies Inc. (Coralville, IA). They were HPLC-purified and freeze-dried by the supplier. The DNA sequences were used as provided and diluted in 10 mM phosphate buffer (PB) solution, pH 7.0,

Received: October 5, 2011 Accepted: December 14, 2011 Published: December 14, 2011 1042

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to give stock solutions of 100 μM. Table 1 depicts the sequences of the oligonucleotides used in this study.

computer (F900 version 6.3 software, Edinburgh Instruments). Absorbance measurements of ABTS•− (ABTS = 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) were performed using a Shimadzu UV-2401PC UV−vis spectrophotometer. Atomic force microscopy (AFM) images were recorded using a Nanoscope 3A controller (Digital Instruments/Veeco Probes, Plainview, NY) with NSC 15 AFM tips (Mikromasch, Tallinn, Estonia) using the tapping mode at their resonant frequency. The DNA sample was deposited on a freshly cleaved mica surface (Structure Probe Inc., West Chester, PA), dried in air, and gently washed with double-distilled water (DDW). Images were analyzed using the WsXM SPIP software (Nanotec, Inc., Madrid, Spain). Analysis of DNA by Colorimetric and Chemiluminescence Measurements. All samples were prepared in 10 mM HEPES (pH 7.0) buffer containing 300 mM NaCl. Each functional hairpin structure (1 μM) was heated to 95 °C for 5 min and then allowed to cool to room temperature (25 °C) for at least 2 h before use. Then different concentrations of the target DNA 3 were incubated with a 0.5 μM mixture of hairpins 1 and 2. For BRCA1 oncogene 7 detection, 0.1 μM hairpin 6 was added to the 0.5 μM mixture of hairpins 1 and 2. For analysis of the G-quadruplex subunits 0.5 μM 4 and 5 were

Table 1. DNA Sequences Used To Construct the Sensing Platform sequence 1 2 3 4 5 6 7

5′ AGG GCG GGT GGG TGT TTA AGT TGG AGA ATT GTA CTT AAA CAC CTT CTT CTT GGG T 3′ 5′ TGG GTC AAT TCT CCA ACT TAA ACT AGA AGA AGG TGT TTA AGT TGG GTA GGG CGG G 3′ 5′ AGA AGA AGG TGT TTA AGT A 3′ 5′ TAC TTA AAC TGG GCG GGT GGG T 3′ 5′ TGG GTC CTT CTT CT 3′ 5′ TTA AAC ACC TTC TTC CAA CAG CTA TAA ACA GTC CTG GAT AAT GGG TTT ATG AAA AAC ACT TTA GAA GAA GGT GTT TAA GTA 3′ 5′ AAA GTG TTT TTC ATA AAC CCA TTA TCC AGG ACT GTT TAT AGC TGT TGG AAG 3′

Instrumentation. Light emission experiments were carried out using a photon-counting spectrometer (FLS 920, Edinburgh Instruments, Livingston, U.K.) equipped with a cooled photomultiplier detection system connected to a

Figure 1. (A) Scheme for the amplified detection of a target DNA 3 using two functional hairpin structures (1 and 2) that upon recognition of the target DNA trigger the autonomous generation of nanowires consisting of the HRP-mimicking DNAzyme. The DNAzyme units act as readout labels for the colorimetric or chemiluminescent detection of the target DNA. (B) AFM image and cross-section analysis of the resulting DNAzyme nanowires. (C) Time-dependent absorbance changes, following ABTS•− at λ = 420 nm, upon analysis of different concentrations of the target DNA 3 using the sensing platform shown in (A): (a) 0 M, (b) 1 × 10−13 M, (c) 1 × 10−12 M, (d) 1 × 10−11 M, (e) 1 × 10−10 M, (f) 1 × 10−9 M, and (g) 1 × 10−8 M target 3 and (h) hemin only, 75 nM. Inset: Calibration curve corresponding to the absorbance changes observed after a fixed time interval of 10 min upon analysis of different concentrations of the target DNA 3. (D) Chemiluminescence spectra corresponding to the analysis of different concentrations of 3, according to the scheme outlined in (A): (a) 0 M, (b) 1 × 10−13 M, (c) 1 × 10−12 M, (d) 1 × 10−11 M, (e) 1 × 10−10 M, (f) 1 × 10−9 M, and (g) 1 × 10−8 M and (h) hemin only, 75 nM. Inset: Calibration curve corresponding to the chemiluminescence intensities at λ = 415 nm in the presence of different concentrations of 3. All systems consist of 1, 500 nM, and 2, 500 nM, in the presence of the respective concentration of 3 in 10 mM HEPES buffer, pH 7.0, that includes 300 mM NaCl. 1043

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incubated with different concentrations of the target DNA 3. All samples were incubated for a fixed time interval of 6 h prior to the measurements. In all experiments a 50 μL fraction was extracted from the samples and used for the absorbance measurements of ABTS•− and the chemiluminescence readout of luminol. Absorbance measurements were conducted in a cuvette that included 50 μL of the samples and 150 μL of a buffer solution that included 75 nM hemin, 2 mM ABTS2−, and 200 μM H2O2. Light emission experiments were carried out in a cuvette that included 50 μL of the samples and 150 μL of a buffer solution that included 75 nM hemin, 0.5 mM luminol, and 30 mM H2O2. For AFM characterization of the DNA wires that were produced by cross-opening of the functional DNA hairpin structures 1 and 2, the concentration of both hairpins was 0.5 μM and that of the target DNA 3 was 10 nM. For this reaction, samples were prepared in 10 mM HEPES with no salt added.

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RESULTS AND DISCUSSION The analytical system is depicted in Figure 1A. It consists of two hairpin structures (1 and 2). Hairpin 1 includes at the 5′ end of the stem region the encoded sequence that consists of three-fourths of the G-quadruplex sequence, domain I, linked to the programmed conserved sequence composed of domains IV and III. The 3′ end of the hairpin consists of sequence II comprising one-fourth of the G-quadruplex sequence. Domain I is partially hybridized with domain III. Sequence II is linked to domain III, which is complementary to parts of domain IV, resulting in a stable hairpin configuration. Hairpin 1 includes the recognition sequence for the target nucleic acid 3. Hairpin 2 is functionalized at its 5′ and 3′ ends with one-fourth of the Gquadruplex and three-fourths of the G-quadruplex sequences, respectively. The three-fourths of the G-quadruplex sequence is linked to the programmed sequences III′ and IV′, which are complementary to domains III and IV, respectively, in hairpin 1. The fact that sequence I is partially hybridized with domain III or IV′ of the stems in hairpins 1 and 2 prevents the selfassembly of the active hemin/G-quadruplex DNAzyme. The opening of hairpin 1 by the target 3 releases the single-stranded domain IV and the conserved three-fourths of the Gquadruplex (domain I), step 1. Domain IV in the resulting structure (a) hybridizes with the stem region of 2, domain IV′, and opens hairpin 2 to yield structure (b), step 2. The resulting structure (b) cross-hybridizes with hairpin 1 by hybridization of domain III′ to region III of the stem of 1, step 3. This results in structure (c), where the G-quadruplex subunits (domains I and II) self-assemble into the G-quadruplex structure.39 By the autonomous cross-opening of hairpins 2 and 1, steps 2 and 3, respectively, polymer wires consisting of the G-quadruplex units are formed, and in the presence of hemin the resulting catalytic hemin/G-quadruplex HRP-mimicking DNAzymes are anticipated to be formed. The hemin/G-quadruplex nanowires are, then, anticipated to catalyze the oxidation of ABTS2− by H2O2 to ABTS•− (λmax = 420 nm) or to catalyze the generation of chemiluminescence of oxidation of luminol by H2O2 (λmax = 415 nm). Figure 1B depicts the AFM image of the polymeric nanowires generated upon interaction of hairpins 1 and 2, 500 nM each, with target 3, 10 nM. Micrometer long nanowires are observed. The height of the nanowires is ca.1.7 nm. It should be noted that many of the nanowires form bundles. This is attributed to the intercrossing of polymer chains by the formation of interchain G-quadruplexes.

Figure 2. (A) Analysis of the target DNA by two subunits that yield the target-induced formation of the hemin/G-quadruplex DNAzyme. (B) Comparison of the time-dependent absorbance changes corresponding to the formation of ABTS•− upon analysis of the target DNA 3, 1 × 10−8 M, using (a) the autonomous cross-opening of two hairpins and the generation of nanowires consisting of the HRPmimicking DNAzyme according to Figure 1A and (b) two subunits generating the HRP-mimicking DNAzyme according to Figure 2A.

Figure 1C shows the time-dependent absorbance changes upon analysis of variable concentrations of the target DNA 3 by the two hairpins 1 and 2. As the concentration of the target is lower, the absorbance changes decrease in their intensities. Control experiments reveal that only minute absorbance changes are observed in the absence of the target 3, Figure 1C, curve a, implying that the absorbance changes, indeed, originate from the cross-opening of hairpins 1 and 2 and the generation of the active hemin/G-quadruplex DNAzyme units. The calibration curve for analyzing 3 is depicted in Figure 1C, inset, demonstrating that 3 could be analyzed with a sensitivity that corresponds to 1 × 10−13 M. Similarly, the hemin/Gquadruplex wires were applied as catalysts for the generation of chemiluminescence. Figure 1D shows the chemiluminescence signals generated by the system upon analysis of variable concentrations of the target 3 according to Figure 1A. In these experiments the chemiluminescence signal was recorded after the autonomous formation of the DNAzyme nanowires, through the cross-opening of hairpins 1 and 2 that was allowed to proceed for a fixed time interval of 6 h. The resulting calibration curve is shown in Figure 1D, inset. The analyte 3 could be detected with a sensitivity corresponding to 1 × 10−13 M. It should be noted that the analysis of 3 according to Figure 1A was performed using a fixed time interval of 6 h. Figure S1, Supporting Information, shows the time-dependent absorbance changes observed upon analysis of 3, 1 × 10−8 M, by the analytical platform depicted in Figure 1A using different time intervals for the polymerization of the hemin/G-quadruplex nanowires. We find that already after 2 h measurable absorbance changes are detected. The absorbance changes are intensified as the polymerization of the hemin/G-quadruplex is prolonged, and they level off to a saturation value after 6 h. Accordingly, the measurements in the present study were 1044

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Figure 4. Time-dependent absorbance changes, following ABTS•− at λ = 420 nm, upon analysis of the diluted DNAzyme nanowires using the sensing platform shown in Figure 1A: (a) no dilution, (b) diluted by a factor of 2, (c) diluted by a factor of 4, (d) diluted by a factor of 5, (e) diluted by a factor of 6. The inset shows the calibration curve corresponding to the absorbance changes observed after dilution of the resulting DNAzyme nanowires triggered by 1 × 10−8 M analyte DNA 3. The system consists of 1, 500 nM, and 2, 500 nM, in the presence of 1 × 10−8 M target DNA 3 in 10 mM HEPES buffer, pH 7.0, that includes 300 mM NaCl.

DNAzyme structure. The time-dependent absorbance changes upon analysis of different concentrations of the target 3 by subunits 4 and 5 using ABTS•− as the readout signal are depicted in Figure S2 (Supporting Information). Similarly, the analysis of different concentrations of the target DNA 3 by subunits 4 and 5 and using chemiluminescence as the readout signal are shown in Figure S3 (Supporting Information). The detection limit for analyzing 3 by the two subunits 4 and 5 using ABTS•− or chemiluminescence as the readout signal corresponds to 1 × 10−10 M. This value is ca. 103 less sensitive as compared to that of the sensing platform depicted in Figure 1A, which involves the target-induced cross-opening of hairpins 1 and 2 and the generation of the hemin/G-quadruplex DNAzyme nanowires. To highlight the amplification effect of the cross-opening of hairpins 1 and 2 on the sensing of 3, we examined the analysis of 3, 1 × 10−8 M, by the two platforms outlined in Figures 1A and 2A, respectively. Figure 2B, curve a, shows the time-dependent absorbance changes due to the formation of ABTS•− upon analysis of 3 according to Figure 1A, whereas Figure 2B, curve b, depicts the analysis of the target DNA 3 by subunits 4 and 5 according to Figure 2A. Evidently, the absorbance changes upon analysis of the DNA 3 by the target-induced cross-opening of hairpins 1 and 2 are ca. 6-fold higher than those observed in the presence of subunits 4 and 5 only. The enhanced absorbance changes upon analysis of 3 by the cross-opening of hairpins 1 and 2 are attributed to the higher content of the active DNAzyme that transduces the sensing events. While in the presence of subunits 4 and 5 the content of the DNAzyme is controlled by the concentration of the analyte (the maximum content of DNAzyme is equivalent to the concentration of the analyte), the target-induced crossopening of hairpins 1 and 2 leads to the formation of numerous DNAzyme units, assembled as nanowires, by a single recognition event. One important aspect of the amplified detection platform shown in Figure 1A relates to the specificity of the system and its ability to discriminate mutants from the analyte. Figure 3A

Figure 3. (A) Sequences of the analyte 3 and the respective mutations. (B) Time-dependent absorbance changes upon analysis of different analytes according to Figure 1A: (a) target DNA 3, 1 × 10−10 M, (b) one-base mutant 3a, 1 × 10−10 M, (c) two-base mutant 3b, 1 × 10−10 M, (d) three-base mutant 3c, 1 × 10−10 M, (e) in the absence of the analyte 3. For detailed experimental conditions see the caption of Figure 1 and the Experimental Section. (C) Chemiluminescence spectra upon analysis of different analytes according to Figure 1A: (a) target DNA 3, 1 × 10−10 M, (b) one-base mutant 3a, 1 × 10−10 M, (c) two-base mutant 3b, 1 × 10−10 M, (d) three-base mutant 3c, 1 × 10−10 M, (e) in the absence of the analyte 3.

performed using a time interval of 6 h for the polymerization of the DNAzyme nanowires. Nonetheless, it is evident from Figure S1, inset, that the polymerization time interval can be shortened to only 4 h with an accompanying decrease in the readout signal that corresponds to 15%. To demonstrate the effect of the cross-opening process of hairpins 1 and 2 on the amplified detection of the target DNA 3, we examined the analysis of 3 by the two DNAzyme subunits 4 and 5 (three-fourths and one-fourth of the G-quadruplex, respectively) according to Figure 2A. In this system the formation of the G-quadruplex DNAzyme is controlled by the concentration of the analyte that cooperatively stabilizes the 1045

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Figure 5. (A) Analysis of the BRCA1 oncogene 7 using the functional hairpins 1 and 2 as amplifying units that generate the DNAzyme nanowires and implanting a helper hairpin 6 that is opened by the analyte and activates the cross-opening of the functional hairpins and the synthesis of the DNAzyme nanowires. (B) Time-dependent absorbance changes, following ABTS•− at λ = 420 nm, upon analysis of different concentrations of the target DNA 7 using the sensing platform shown in (A): (a) 0 M, (b) 1 × 10−13 M, (c) 1 × 10−12 M, (d) 1 × 10−11 M, (e) 1 × 10−10 M, (f) 1 × 10−9 M, (g) 1 × 10−8 M, (h) 1 × 10−7 M, and (i) 1 × 10−6 M target 7 and (j) 1 × 10−6 M 7 in the absence of 6. Inset: Calibration curve corresponding to the absorbance changes observed after a fixed time interval of 10 min upon analysis of different concentrations of the target DNA 7. (C) Chemiluminescence spectra corresponding to the analysis of different concentrations of 7 according to the scheme outlined in (A): (a) 0 M, (b) 1 × 10−13 M, (c) 1 × 10−12 M, (d) 1 × 10−11 M, (e) 1 × 10−10 M, (f) 1 × 10−9 M, (g) 1 × 10−8 M, (h) 1 × 10−7 M, and (i) 1 × 10−6 M target 7 and (j) 1 × 10−6 M 7 in the absence of 6. Inset: Calibration curve corresponding to the chemiluminescence intensities at λ = 415 nm in the presence of different concentrations of 7. All systems consist of 1, 500 nM, 2, 500 nM, and 6, 100 nM, in the presence of the respective concentration of 7 in 10 mM HEPES buffer, pH 7.0, that includes 300 mM NaCl.

Table 2. Enzyme- and DNAzyme-Amplified Detection of DNA system carbolic regeneration of analyte by DNAzyme detection of DNA by DNAzyme cascade replication/nicking DNA machinery Rolling circle amplification (RCA) and DNAzyme machinery recycling of analyte autonomous assembly of HRP-mimicking DNAzyme nanowires

catalyst

sensing duration (h)

sensitivity (M)

ref

Mg2+-dependent DNAzyme Mg2+-dependent DNAzyme; HRP-mimicking DNAzyme polymerase/nicking enzyme; HRP-mimicking DNAzyme polymerase; HRP-mimicking DNAzyme

12 7

1 × 10−12 1 × 10−12

26 30

1.5

1 × 10−14

36

2

1 × 10−14

38

exonuclease III HRP-mimicking DNAzyme

2 4−6 h

2 × 10−9 1 × 10−13

40 present study

mismatched analytes 3b and 3c show spectral changes that are almost identical to the background signal of the system. Also, the single-base mismatched analyte 3a can be easily discriminated from the target DNA 3, implying that the system reveals high selectivity. Similarly, Figure 3C shows the chemiluminescence spectra generated by the analytical platform shown in Figure 1A upon analysis of the target DNA 3, 1 × 10−10 M, and the mutants 3a, 3b, and 3c. One may realize that the chemiluminescence intensities generated by the different mutants are very close to the background chemiluminescence intensity of the system (in the absence of any analyte). These

shows the sequence of the target analyte 3 and the sequences of the one-, two-, and three-base mutations 3a, 3b, and 3c, respectively, that were examined to elucidate the specificity of the sensing system. Figure 3B shows the time-dependent absorbance changes of ABTS•− upon analysis of the target DNA 3, 1 × 10−10 M, curve a, and upon the analysis of 3a, curve b, 3b, curve c, and 3c, curve d, all at a concentration corresponding to 1 × 10−10 M. The time-dependent absorbance change in the absence of any of the analytes is shown in Figure 3B, curve e. This curve represents the background absorbance of the system. One may realize that the two-base and three-base 1046

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selectivity and are cost-effective in comparison with the enzymatic assays.

results further demonstrate the capability of the analytical platform to discriminate the mutants from the analyte structure. We have further examined the possibility to implement the sensing platform under real sample preparation conditions (see the results and discussion in the Supporting Information, Figure S4). One further aspect that needs to be addressed relates to the effect of bundling of the G-quadruplex nanowires on the resulting activity of the self-assembled hemin/G-quadruplex DNAzymes and on the resulting absorbance changes and chemiluminescence intensities. As pointed out, the AFM images of the resulting G-quadruplex nanowires form, in addition to simple singular DNA wires, also bundles of wires. The formation of these bundles was attributed to the interchain formation of G-quadruplexes. One may argue that the interchain complexes may differ in their catalytic activities, thus influencing the resulting absorbance/chemiluminescence changes upon analysis of 3. To resolve this issue, we analyzed the target DNA 3, 1 × 10−8 M, according to Figure 1A, and prior to the addition of ABTS 2− /H 2 O 2 the resulting polymerized DNA nanowire solution was diluted and incubated for a 1/2 h. We expect that upon dilution the formation of individual hemin/G-quadruplex nanowires will be favored. Figure 4 shows the time-dependent absorbance changes upon analysis of the different diluted hemin/G-quadruplex nanowire solutions. One may observe a linear dependence, implying that the hemin/G-quadruplexes in the individual nanowires or in the bundled nanostructures exhibit similar activities. The enzyme-free sensing platform depicted in Figure 1A requires structural optimization of the hairpins, so that random cross-opening is eliminated (see Figure S5, Supporting Information). This optimized system can be used, however, as a versatile amplified sensing platform for any target DNA. This was achieved by introducing a “helper” hairpin structure (6), which opens in the presence of the analyte DNA and yields a single-strand tether of the stem region (III′). This region corresponds to the sequence of 3 that activates the crossopening of hairpins 1 and 2, Figure 5A. This was exemplified with the analysis of the BRCA1 oncogene 7. Opening of hairpin 6 by 7 activates the autonomous cross-opening of the two hairpins 1 and 2 and the formation of the hemin/G-quadruplex DNAzyme nanowires. Parts B and C of Figure 5 show the timedependent absorbance changes (due to the formation of ABTS•−) and the chemiluminescence intensities upon analysis of different concentrations of the oncogene. The resulting calibration curves are shown in Figures 5B,C insets. The oncogene could be analyzed with a sensitivity that corresponded to 1 × 10−13 M. In conclusion, the present study has introduced a new enzyme-free amplified detection platform of DNA. The system involved the use of two functional hairpin structures that upon recognition of the target DNA activate an autonomous crossopening process that leads to the formation of hemin/Gquadruplex DNAzyme wires that provide the colorimetric or chemiluminescence readout of the sensing process. The system reveals impressive selectivity, and one-, two-, and three-base mutants could be discriminated from the analyte by the analytical platform. Table 2 summarizes different amplified DNA sensing platforms that implement protein-based enzymes or catalytic nucleic acids (DNAzymes) as amplifying catalysts. The protein-based catalytic systems reveal a ca. 10-foldenhanced sensitivity and a ca. 2-fold faster analysis time interval. In contrast, the DNAzyme-based systems exhibit high

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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

*E-mail: [email protected]. Phone: 972-2-6585272. Fax: 972-2-6527715.

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ACKNOWLEDGMENTS This study was supported by the FP7 EU ECCell project. The first two authors contributed equally to this work. REFERENCES

(1) Patel, P. D. Trends Anal. Chem. 2002, 21, 96−115. (2) Bagni, G.; Osella, D.; Sturchio, E.; Mascini, M. Anal. Chim. Acta 2006, 573, 81−89. (3) Wang, J. Biosens. Bioelectron. 2006, 21, 1887−1892. (4) Bond, J. W. J. Forensic Sci. 2007, 52, 128−136. (5) Ho, H.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168− 178. (6) Duan, X.; Liu, L.; Feng, F.; Wang, S. Acc. Chem. Res. 2010, 43, 260−270. (7) Iliuk, A. B.; Hu, L.; Tao, W. A. Anal. Chem. 2011, 83, 4440−4452. (8) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192−1199. (9) Wang, J.; Liu, G.; Merkoçi, A. J. Am. Chem. Soc. 2003, 125, 3214− 3215. (10) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134−9137. (11) Fojta, M.; Havran, L.; Vojtiskova, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532−6533. (12) Li, D.; Yan, Y.; Wieckowska, A.; Willner, I. Chem. Commun. 2007, 3544−3546. (13) Weizmann, Y.; Patolsky, F.; Willner, I. Analyst 2001, 126, 1502− 1504. (14) Lucarelli, F.; Tombelli, S.; Minunni, M.; Marrazza, G.; Mascini, M. Anal. Chim. Acta 2008, 609, 139−159. (15) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769−774. (16) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770−772. (17) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268−2271. (18) Golub, E.; Pelossof, G.; Freeman, R.; Zhang, H.; Willner, I. Anal. Chem. 2009, 81, 9291−9298. (19) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071−9077. (20) Wang, J.; Zhou, H. S. Anal. Chem. 2008, 80, 7174−7178. (21) Breaker, R. R. Nat. Biotechnol. 1999, 17, 422−423. (22) Famulok, M.; Mayer, G.; Blind, M. Acc. Chem. Res. 2000, 33, 591−599. (23) Kolpashchikov, D. M. ChemBioChem 2007, 8, 2039−2042. (24) Silverman, S. K. Chem. Commun. 2008, 3467−3485. (25) Sando, S.; Sasaki, T.; Kanatani, K.; Aoyama, Y. J. Am. Chem. Soc. 2003, 125, 15720−15721. (26) Wang, F.; Elbaz, J.; Teller, C.; Willner, I. Angew. Chem., Int. Ed. 2011, 50, 295−299. (27) Travascio, P.; Li, Y. F.; Sen, D. Chem. Biol. 1998, 5, 505−517. (28) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430−7431. (29) Teller, C.; Shimron, S.; Willner, I. Anal. Chem. 2009, 81, 9114− 9119. 1047

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(30) Elbaz, J.; Moshe, M.; Shlyahovsky, B.; Willner, I. Chem.Eur. J. 2009, 15, 3411−3418. (31) Niazov, T.; Pavlov, V.; Xiao, Y.; Gill, R.; Willner, I. Nano Lett. 2004, 4, 1683−1687. (32) Freeman, R.; Liu, X.; Willner, I. J. Am. Chem. Soc. 2011, 133, 11597−11604. (33) Liu, X.; Freeman, R.; Golub, E.; Willner, I. ACS Nano 2011, 5, 7648−7655. (34) Pelossof, G.; Tel-Vered, R.; Elbaz, J.; Willner, I. Anal. Chem. 2010, 82, 4396−4402. (35) Pelossof, G.; Tel-Vered, R.; Liu, X.; Willner, I. Chem.Eur. J. 2011, 17, 8904−8912. (36) Weizmann, Y.; Beissenhirtz, M. K.; Cheglakov, Z.; Nowarski, R.; Kotler, M.; Willner, I. Angew. Chem., Int. Ed. 2006, 45, 7384−7388. (37) Tian, Y.; He, Y.; Mao, C. ChemBioChem 2006, 7, 1862−1864. (38) Cheglakov, Z.; Weizmann, Y.; Basnar, B.; Willner, I. Org. Biomol. Chem. 2007, 5, 223−225. (39) Deng, M.; Zhang, D.; Zhou, Y.; Zhou, X. J. Am. Chem. Soc. 2008, 130, 13095−13102. (40) Hsieh, K.; Xiao, Y.; Tom, S. H. Langmuir 2010, 26, 10392− 10396.

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dx.doi.org/10.1021/ac202643y | Anal. Chem. 2012, 84, 1042−1048