Label-Free Colorimetric Aptasensor Based on Nicking Enzyme

Mar 28, 2013 - Label-Free Colorimetric Aptasensor Based on Nicking Enzyme Assisted Signal Amplification and DNAzyme Amplification for Highly Sensitive...
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Label-Free Colorimetric Aptasensor Based on Nicking Enzyme Assisted Signal Amplification and DNAzyme Amplification for Highly Sensitive Detection of Protein Yong Huang,§ Jia Chen,§ Shulin Zhao,* Ming Shi, Zhen-Feng Chen, and Hong Liang* Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin, 541004, China S Supporting Information *

ABSTRACT: Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. Here, we develped a novel label-free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein. The system consists of a hairpin DNA probe carrying an aptamer sequence for target, a G-riched DNA probe containing two G-riched DNAzyme segments and the recognition sequence as well as cleavage site for nicking enzyme, a blocker DNA, and the nicking enzyme. The hybridization of the G-riched DNA with the blocker DNA prohibits the formation of the activated DNAzymes in the absence of target. Upon addition of target to the system, the hairpin probe is opened by the specific recognition of the target to its aptamer. The open hairpin probe hybridizes with a G-riched DNA and forms a DNA duplex, which triggers the selective cleavage of the G-riched DNA probe by nicking enzyme, leading to the release of the aptamer−target complex and the G-riched DNAzyme segments. The released open hairpin probe then hybridizes with another G-riched DNA probe, and the cycle starts anew, resulting in the continuous cleavage of the G-riched DNA probes, generating a much of G-riched DNAzyme segments. The G-riched DNAzyme segments interact with hemin and generates the activated DNAzyme that can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS•−, thus providing the amplified colorimetric detection of target. With the use of thrombin (Tb) as a proof-of-principle analyte, this sensing platform can detect Tb specifically with a detection limit as low as 1.5 pM, which is at least 4 orders of magnitude lower over the unamplified colorimetric assay. Moreover, the assay does not involve any chemical modification of DNA, which is simple and low-cost. This sensing platform provides a promising approach for the amplified analysis of target molecules.

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labels,23 and the use of nucleases as biocatalytic amplifiers.24,25 The improved sensitivities of homogeneous aptasensors were demonstrated by the different amplification techniques mentioned above. However, most of these amplified aptasensors relied on the chemical modification of aptamers or signaling DNAs for the labeling of fluorophores and quenchers, or nanoparticles, which was rather costly, timeconsuming, and sophisticated. Therefore, the development of label-free homogeneous aptasensors that is simpler and more sensitive is still required. Simple colorimetric methods have attracted significant interest due to its rapidness, simplicity, and no need to use expensive analytical instruments. In recent years, many aptamer-based colorimetric sensors have been developed.26−34 However, most of these colorimetric methods need steps such as modifying aptamers onto the gold nanoparticles (AuNPs) and separating the modified AuNPs from the unmodified

ptamers hold great promise for the biosensing of lowmolecular-weight substrates or macromolecules due to their relative ease of isolation and modification, high specificity, and good stability.1−3 Numerous optical,4−6 and electrochemical,7−9 aptasensors have been developed in recent years. Among them, the homogeneous optical aptasensors have been particularly popular due to its simplicity and rapidness.10,11 To date, a number of homogeneous aptasensors based on fluorescence12−14 and ultraviolet absorbance15,16 have been developed for the sensing of various targets. Albeit substantial progress was accomplished, a major limitation of homogeneous optical aptasensors is their relatively low-affinity binding constants of the analytes to the respective aptamers that lead to low assay sensitivities.17 Recently, several intriguing amplification techniques have been implemented in the development of homogeneous aptasensors for overcoming the above-mentioned problem. These techniques include the use of autonomous aptamer-based machine,18 rolling circle amplification process,19,20 isothermal circular strand-displacement polymerization,21 target-catalyzed hairpin assembly process,22 the application of silica nanoparticles as amplifying © 2013 American Chemical Society

Received: December 22, 2012 Accepted: March 28, 2013 Published: March 28, 2013 4423

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AuNPs or surplus aptamers. These steps, not only led to complication and relatively high cost of the experiments, but labeling also could weaken the affinity between the target and the aptamer. Therefore, developing unmodified and label-free colorimetric aptasensors to simplify the detection process would be important and attractive. Nicking endonucleases are a special family of restriction endonucleases that can recognize a specific sequence of a double-stranded DNA (dsDNA) and cut one strand of a dsDNA.35,36 This function has been used to develop different nicking enzyme based amplified detection platforms for biosensing of different analytes.37−39 Some techniques based on nicking enzyme are demonstrated for development of homogeneous fluorescence or colorimetric aptasensors.40,41 However, most of the current nicking enzyme based optical aptasensors require the additional modification of aptamer probes or signaling DNA probes. DNAzyme is a class of catalytic nucleic acids originated from an in vitro selection process termed the systematic evolution of ligands by exponential enrichment (SELEX).42 A hemin/G-quadruplex horseradish peroxidase (HRP) mimicking DNAzyme can catalyze the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonate disodium salt (ABTS2−) by H2O2 to the colored product ABTS•− 43 or enhance the chemiluminescence of the luminol−H2O2 system.44 These properties of HRPmimicking DNAzyme have been extensively used for biosensing events. For example, the hybridization of the target DNA with a molecular beacon or the recognition of the analyte by its hairpin aptamer led to the activation of DNAzyme for optical detection of DNA or aptamer substrates.45,46 Similarly, thymine-rich functionalized DNA or cytosine-rich functionalized DNA coupling with the HRP-mimicking DNAzyme was utilized to amplify Hg2+ or Ag+ ion detection.47,48 Also, the activation of DNAzyme by the target-induced conjugation of two split aptamer fragments or segmented DNA elements to optical detection of aptamer substrates or DNAs was reported.49 Although these techniques can be quite powerful, higher sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when target molecule density is extremely low. In the present work, taking advantage of nicking enzyme signaling amplification (NESA) and HRP-mimicking DNAzyme amplification, we designed a label-free colorimetric method for amplified detection of proteins. This method does not need any chemical modification of DNA probe for labeling, the target molecule can be simply detected by the naked eye or with UV−vis spectroscopy, which is simple and cost-effective. With the use of thrombin (Tb) as a proof-ofprinciple analyte, this sensing platform exhibited high sensitivity and specificity toward Tb versus other nontargeted proteins. The proposed sensing platform promises a powerful approach for amplified analysis of target molecules.

Table 1. DNA Oligonucleotides Sequence Used in This Work oligonucleotide name hairpin DNA probe G-riched DNA probe blocker DNA hairpin DNA-2 hairpin DNA-3

sequence (5′ to 3′) description AGT CCG TGG TAG GGC AGG TTG GGG TGA CTG CTG A T TTT TTG AGC CTC AGC AGT CAC C CAC AGG GTT GGG CGG GAT GGG TGC TG GGT GAC TGC↓T GAG GCT GGG GTA GGG CGG GTT GGG AAT T CCC TTA AGT GTC CC T GAC GAG TGC TGT AAG GTA CCG TCT TCT CAG CCT CAG CAC TCG TCA TC ACT ATG CTG GTT CCG TCT TTT TTT CCT CAG CAT AGT GA

a

The italic bold letters of a hairpin DNA probe is the aptamer sequences of Tb, the underlined letters composed of the hairpin DNA probe stem region. The blue letters of a hairpin DNA probe and a Griched DNA probe are completely complementary, which contain the recognition sequences for Nb.BbvCI. The arrow indicates the nicking position.

albumin (HSA), fibrinogen (FIB), hemoglobin (Hb), myoglobin (Mb), zonolysin, glucose (GLU), lactic acid, hemin, ethidium bromide (EB), Triton X-100, disodium 2,2′-azinobis (3-ethylbenzothiazoline)-6-sulfonate (ABTS), H2O2, sodium 4-(2-hydroxyethyl) piperazine-1-ethanesulfonate (HEPES), [tris(hydroxymethy-l)aminomethane] (Tris), and standardized human serum were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Human Immunoglobulin G (IgG) was purchased from Beijing Biosynthesis Biotechnology Co. (Beijing, China). The nicking enzyme Nb.BbvCI and 10 × NEB buffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, and 10 mM dithiothreitol, pH 7.9) were purchased from the New England Biolabs, Inc. (Ipswich, MA, USA). Agarose G-10 was obtained from Biowest. Ultra low range molecular weight DNA ladder was purchased from Fermentas, Inc. (Vilnius, Lithuania). Five × TBE buffer (pH 7.9) was provided by Sangon Biotechnology Co. Ltd. (Shanghai, China). All other reagents were of analytical grade and were used without further purification. Water was purified with a Milli-Q plus 185 equip from Millipore (Bedford, MA, USA) and used throughout the work. Absorbance Measurements. Absorbance measurements were performed using a TU-1901 UV−visible spectrophotometer (Beijing Purkinje General Instrument Co, Ltd., China) with a 0.5 cm path length quartz cuvette. The absorption spectra of the solution were measured in the wavelength range from 900 to 400 nm at a fixed time interval of 5 min. The rate of the peroxidase-mimicking reaction was monitored at 418 nm. Procedure for Thrombin Assay. The detailed procedure for Tb detection was as follows. First, a G-riched DNA probe (25 μL, 10 μM) and blocker DNA (30 μL, 10 μM) were mixed and incubated at room temperature for 1 h. Second, 5 μL of 20 mM Tris-HCl solution that contained 10 μM of the hairpin probe and 5 μL varying concentrations of Tb or other proteins was added the above mixture and incubated for another 1 h at 37 °C. Then, Nb.BbvCI (5 μL, 6 u/μL) and 2 × NEB buffer 2 (70 μL) were added and allowed to incubate for 2 h at 37 °C. After incubation, the solution was diluted with 300 μL HEPES buffer solution (pH 8.0, 25 mM HEPES, 0.05% (w/v) Triton X-100, 1% (v/v) DMSO, 20 mM KCl, 200 mM NaCl). Subsequently, hemin (5 μL) was added to obtain the mixture

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EXPERIMENTAL SECTION Materials and Reagents. Oligonucleotides used in this work were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and their sequences were listed in Table 1. The oligonucleotides were used as provided and diluted in pH 8.0, 20 mM Tris-HCl buffer solution (containing 100 mM NaCl, 20 mM KCl, and 2 mM MgCl2) to give stock solutions of 10 μM. And each oligonucleotide was heated to 95 °C for 10 min, and slowly cooled down to room temperature before use. Tb, bovine serum albumin (BSA), human serum 4424

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and give the final concentration of 0.6 μM hemin and incubated for 1 h at room temperature to form the hemin/G-quadruplex structures. Finally, the ABTS (50 μL) and H2O2 (5 μL) substrates were added to allow the biocatalyzed oxidation of ABTS2− and the product solution to yield a total volume of 500 μL, the final concentrations of ABTS and H2O2 were 2 and 2 mM, respectively. The resulting samples were tested with a UV−vis spectrometer. Gel Electrophoresis. Gel electrophoresis was used to confirm the hairpin probe−Tb binding and nicking enzyme Nb.BbvCI-induced strand-scission reaction. Samples for gel electrophoresis assays were prepared as follows: (1) G-riched DNA probe (2.5 μM) was used as sample one; (2) hairpin DNA probe (0.5 μM) was used as sample two; (3) blocker DNA (3 μM) was used as sample three; (4) the mixture of Griched DNA probe (2.5 μM) and blocker DNA (3 μM) was incubated at room temperature for 1 h, and used as sample four; (5) the mixture of hairpin DNA probe (0.5 μM) and Tb (2.5 nM) was incubated for 1 h at 37 °C, and used as sample five; (6) the mixture of the hairpin DNA probe (0.5 μM) and blocker DNA (3 μM) was used as sample six; (7) sample was prepared by incubating the mixture of G-riched DNA probe (2.5 μM) and blocker DNA (3 μM) at room temperature for 1 h, and then added hairpin DNA probe (0.5 μM) and Tb (2.5 nM) to the mixture for incubating another 1 h at 37 °C; (8) sample was prepared similarly mentioned above procedure for Tb assay (without addition of hemin, ABTS and H2O2), but with the following changes: 2.5 μM G-riched DNA probe, 3 μM blocker DNA, 0.5 μM hairpin DNA probe, 2.5 nM Tb, 150 units Nb.BbvCI. The gel electrophoresis experiments were performed by using a DYCP-31A Electrophoresis Cell (Beijing LiuYi Instrument Factory, China) equipped with DYY-5 Electrophoresis Power Supply (Beijing LiuYi Instrument Factory, China). In the gel electrophoresis assay, each prepared sample (8 μL) was put on 5% agarose gels to separate the related substances. The electrophoresis was carried in 0.5 × TBE buffer (pH 7.9) at 120 V constant voltage for 1 h. After EB staining, the gels were scanned using the Omega 16ic Gel imaging system (ULTRA-LUM, USA). Circular Dichroism Experiments. Three samples used for circular dichroism (CD) assays were shown as follows: sample one was prepared by incubating the mixture of G-riched DNA probe (1 μM) and blocker DNA (1 μM) at room temperature for 1 h, and then hairpin DNA probe (1 μM) was added to the mixture for incubating another 1 h at 37 °C; sample two was prepared by incubating the mixture of G-riched DNA probe (1 μM) and blocker DNA (1 μM) at room temperature for 1 h, and then hairpin DNA probe (1 μM) and Tb (1 μM) were added to the mixture for incubating another 1 h at 37 °C; sample three was prepared by adding 60 U Nb.BbvCI into the mixture prepared in sample two, and then the resulting mixture was incubated for 2 h at 37 °C. Sample (500 μL each) assays were performed by using J-810 Circular Dichroism spectrophotometer (JASCO, Japan). CD spectra were measured at room temperature using a quartz cell with 1 cm path length, CD spectra were collected from 230 to 350 nm with a canning speed of 200 nm/min. The bandwidth was 2 nm, the data pitch was 1 nm, and the response time was 2 s. All experiments were repeated three times. Preparation of Human Serum Samples. The standardized human serum samples were diluted with 1 × NEB buffer 2 in the ratios of 1:20, 1:40, 1:60, 1:80, and 1: 100, respectively. After that, all these diluted serum samples were

analyzed in a similar way to that of Tb detection, and the signal responses were compared with those from the buffer solution (control). In addition, the 60-fold dilution of standardized human serum was spiked at different concentrations of Tb (6.15, 61.5, and 615 pM) and analyzed to evaluate the potential application of the present method.

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RESULTS AND DISCUSSION Principle of NESA and DNAzyme Amplification Based Colorimetric Aptasensor. The working principle of the designed NESA and DNAzyme amplification based colorimetric aptasensor is illustrated in Scheme 1. The system mainly Scheme 1. Schematic Illustration of the NESA and DNAzyme Amplification Based Colorimetric Aptasensor for Ptotein Assay

consists of a hairpin DNA probe, a G-riched DNA probe, a blocker DNA, and the Nb.BbvCI biocatalyst. The hairpin probe includes three domains, a sequence that is complementary to the G-riched DNA probe and also contains the Nb.BbvCI recognition sequence (yellow, domain I), a single-stranded loop (green, domain II), and the aptamer sequence (purple, domain III) for Tb (as a model analyte). The G-riched DNA probe includes two G-riched DNAzyme segments (blue) and the recognition sequence and cleavage site (orange) for Nb.BbvCI. The blocker DNA is designed to be partly complementary to both ends of the G-riched DNA probe. In the absence of Tb, the blocker DNA hybridizes with the G-riched DNA probe, and forms a quasi-circular structure. As a result, the G-riched DNAzyme segment is prohibited to combine with the hemin molecule and fold into the active hemin/G-quadruplex HRPmimicking DNAzyme structure. When the target Tb is introduced into the system, the hairpin structure is opened by the specific binding of Tb with its aptamer (domain III), thus faciliating the hybridization between the open hairpin probe and the quasi-circular DNA structure to form a DNA duplex. The formation of the DNA duplex triggers the selective enzymatic cleavage of the quasi-circular DNA by Nb.BbvCI, resulting in the release of the G-riched DNAzyme segments and the open hairpin probe. The released open hairpin probe then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure, liberating the G-riched DNAzyme segments and the open hairpin probe. Eventually, each open hairpin probe can go through many cycles, resulting in the digestion of many quasi-circular DNA 4425

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structures, generating many G-riched DNAzyme segments. The G-riched DNAzyme segment products assembles with hemin to form the hemin/G-quadruplexes that exhibit peroxidase-like activity which can oxidize ABTS2− to the colored product ABTS•− (λmax = 418 nm) by H2O2. Thus, we could easily detect the target by the naked eye or with UV−vis spectroscopy. Remarkably, the use of NESA coupled DNAzyme amplification techniques provides a significant amplification of detection signal, which improves substantially the sensitivity of detection compared with traditional colorimetric analysis. Moreover, the proposed assay does not involve any chemical modification, which is simple and cost-effective. Feasibility Study. To verify the feasibility of the proposed assay strategy, the UV−vis absorption spectra under different conditions were investigated. As shown in Figure 1, the UV−vis

Figure 2. Agarose gel electrophoresis analysis: (M) DNA marker; (1) G-riched DNA probe only; (2) hairpin DNA probe only; (3) blocker DNA only; (4) mixture of G-riched DNA probe/blocker DNA; (5) hairpin DNA probe/Tb complex; (6) hairpin DNA probe/G-riched DNA probe/blocker DNA tripartite complex; (7) hairpin DNA probe/G-riched DNA probe/blocker DNA/Tb; (8) hairpin DNA probe/G-riched DNA probe/blocker DNA/Tb/Nb.BbvCl.

displayed a band for the G-riched DNA probe/blocker DNA mixture at almost the same migration position as that in lane 1 due to the short sequences of the blocker DNA. In the presence of Tb (lane 5), there was also a little brighter band at the same migration position as that in lane 2. Lane 6 represents the mixture of the hairpin probe/G-riched DNA probe/blocker DNA, there were two bands at the same migration position as that in lane 1 and lane 2, respectively, indicating no interaction between the hairpin probe and the G-riched DNA probeblocker DNA complex (the quasi-circular DNA structure). When Tb was added to the mixture of the hairpin probe/Griched DNA probe/blocker DNA, the band from the hairpin probe-Tb complex disappeared, and a new band with lower mobility was observed (lane 7), indicating the formation of the hairpin probe-Tb/G-riched DNA probe/blocker DNA tripartite complexes. Compared with lane 7, two new fast migration bands appeared and the band from the hairpin probe-Tb/Griched DNA probe/blocker DNA tripartite complexes disappeared in the lane 8, which demonstrated that a strandscission reaction favored the generation of G-riched DNAzyme segments and the release of the hairpin probe−Tb complex. These results depended on the presence of Tb and strandscission reaction of nicking enzyme Nb.BbvCI. CD Characterization. Evidence for the hairpin probe H1− thrombin binding and occurrence of strand-scission reaction was provided by CD analysis (Supporting Information Figure S1). As can be seen from Figure S1, the CD spectrum of the mixture of the hairpin DNA probe and G-riched DNA probe/ blocker DNA had a negative peak at 240 nm, a small positive peak around 260 nm, and a positive peak at 280 nm (curve 1). It is well-documented that the peaks at 240 and 280 nm are characteristic from double-stranded DNA,50 and a negative peak at 240 nm along with a small positive peak at 260 nm are characteristic for a parallel G-quadruplex system without Nb.BbvCl and Tb.51,52 After interacting with Tb, the intensity of the peak around 260 nm increased, and the intensity of the peak 240 nm decreased (curve 2), meanwhile the peak at 280 nm increased in size. This was caused by the formation of a new parallel G-quadruplex from the biding of the hairpin DNA probe with Tb. Further, because of the formation of the tripartite complexes, the DNA double helix became longer, and thus, the peak intensity at 260 nm was increased. Upon adding more Nb.BbvCl to the system, the peak intensity continued to

Figure 1. UV−vis absorption spectra of sample solutions under different conditions: (1) G-riched DNA probe (0.5 μM)/blocker DNA (0.6 μM) + hairpin DNA probe (0.1 μM); (2) G-riched DNA probe (0.5 μM)/blocker DNA (0.6 μM) + hairpin DNA probe (0.1 μM) + Nb.BbvCI (30 U); (3) G-riched DNA probe (0.5 μM)/blocker DNA (0.6 μM) + hairpin DNA probe (0.1 μM) + Tb (6.1 nM); (4) Griched DNA probe (0.5 μM)/blocker DNA(0.6 μM) + hairpin DNA probe (0.1 μM) + Tb (6.1 nM) + Nb.BbvCI (30 U).

absorbance intensity (at 418 nm) of the mixture of hairpin probe, and the G-riched DNA probe/blocker DNA without (curve 1) and with (curve 2) Nb.BbvCI was relatively low. After addition of 6.1 nM Tb to the above mixture without Nb.BbvCI, the absorbance intensity at 418 nm increased was only 68% (curve 3). However, when both Tb (6.1 nM) and Nb.BbvCI were present in the solution, we observed 947% of signal increase in the absorbance intensity at 418 nm (curve 4). This demonstrated that the absorbance enhancement was attributed to the Nb.BbvCI activity. Thus, the proposed assay strategy could be used for amplified detection of Tb. Gel Electrophoresis Characterization. The viability of proposed strategy was further investigated by gel electrophoresis. The results are shown in Figure 2. The first five lanes showed the G-riched DNA probe, the hairpin probe, blocker DNA, mixture of the G-riched DNA probe/blocker DNA, and the hairpin probe/Tb, respectively. A fast migration band appeared in lane 2 due to the stem helix of the hairpin-probe, while no bands were observed in lane 3 due to the singlestranded structure of the blocker DNA. Lane 1 was the Griched DNA probe, which had a relatively low mobility. Lane 4 4426

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of ABTS2− by H2O2, pH 8.0 was selected for the hairpin probe−Tb binding. Thrombin Detection with the Proposed Colorimetric Aptasensor. To confirm the ability of the designed colorimetric aptasensor to detect target protein, a series of different concentrations of Tb were measured. Figure 4 depicts

increase while the peak at 240 nm decreased further (curve 3). Also, the peak at 280 nm became too weak and disappeared. This was because Nb.BbvCI was able to break up the double helix formed between the hairpin DNA probe−Tb complex and G-riched DNA probe/blocker DNA and the G-riched DNA probe/blocker DNA structure releasing G-riched DNAzyme segments and thus forming more parallel G-quadruplex. Optimization of Assay Conditions. With a fixed design strategy, the performance of the developed aptamer-based colorimetric assay is still strongly influenced by the assay conditions such as the ratio of the blocker DNA and G-riched DNA probe, the hairpin probe−Tb binding time, the cleavage reaction time, and pH value for the hairpin probe−Tb binding. Different assay conditions were investigated in our studies, and it revealed that of most importance were the ratio of the blocker DNA, G-riched DNA probe, the cleavage reaction time, and pH value for the hairpin probe−Tb binding. Figure 3

Figure 4. Detection of Tb in the mixed samples. The absorption intensity within 300 s of the oxygenation products ABTS•− in buffer which consisted of 25 mM HEPES−NH4OH (pH 8.0), 0.05% (w/v) Triton X-100, 1% (v/v) DMSO, 20 mM KCl, 200 mM NaCl. The reaction system also includes 0.5 μM G-riched DNA probe, 0.6 μM blocker DNA, 0.1 μM hairpin DNA probe, 0.6 μM hemin, 2 mM ABTS, and 2 mM H2O2. The concentration of Tb: (a) 0 pM; (b) 2.5 pM; (c) 6.1 pM; (d) 12.3 pM; (e) 24.6 pM; (f) 61.5 pM; (g) 123 pM; (h) 246 pM; (i) 615 pM; (j) 2.5 nM; (k) 6.1 nM.

time-dependent absorbance changes upon analyzing different concentrations of Tb under the optimized conditions. As higher concentrations of Tb were added, the absorbance values increased. This is consistent with the fact that the more active G-riched DNAzyme segments are generated by Tb-assisted nicking enzyme catalyzed DNA cleavge reaction, the higher the amounts of ABTS•− get. Figure 5 depicts the calibration curve of absorption intensity at 418 nm versus the log target

Figure 3. Absorption intensity under the different ratio of the blocker DNA to G-riched DNA probe into the sensing system.

shows the response of the colorimetric aptasensor as a function of the ratio of the blocker DNA and G-riched DNA probe. As the ratio of the blocker DNA and G-riched DNA probe was larger, the relative absorbance was intensified and kept constant after the ratio was 1.2. Thus, the ratio of 1.2 for the blocker DNA and G-riched DNA probe was selected for subsequent studies. The hairpin probe−Tb complex bound to the quasicircular DNA structure mediated the cleavage reaction by nicking enzyme Nb.BbvCI in aqueous solution that generated many G-riched DNAzyme segments, enabling the interaction of a number of hemin molecules for UV−vis absorbance detection. A long cleavage reaction time is then expected to yield enhanced signal amplification. The experimental results indicated the absorbance intensity increased with the increase of cleavage reaction time. By weighing both the sensitivity and the total assay time, the cleavage reaction time of 2 h was selected for the following experiments. The pH value for the hairpin probe−Tb binding was also investigated. As shown in Supporting Information Figure S2, the absorbance intensity increased with the increase of the pH value for the hairpin probe−Tb binding and experienced almost no change between pH 7.4 and 8.0. However, with further increase the pH value, the absorbance intensity decreased. To fit the pH value for the final reaction of HRP-mimicking DNAzyme-catalyzed oxidation

Figure 5. Calibration curve of absorption intensity changes at 418 nm as a function of the log concentration of Tb. 4427

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biological matrixes, the analysis of standardized human serum was conducted by the developed NESA and DNAzyme amplification based colorimetric aptasensor. First, the tolerance of the present method under the different dilution rates of human serum was investigated by analyzing different dilution ratios (1:20, 1:40, 1:60, 1:80, 1:100) of human serum without Tb. The results are shown in Figure 7A. It was clear that the

concentration. As can be seen, a good linearity between the aborsorption intensity and the log concentrations of Tb from 2.5 pM and 6.2 nM was obtained. The regression equation is I = 0.4227log C + 5.2274 with a correlation coefficient of 0.9976, where I and C represent the absorption intensity and the Tb concentration, respectively. The limit of detection was estimated to be 1.5 pM from three times the standard deviation corresponding to the blank sample detection. Supporting Information Table S1 compares the performance of the proposed method to some previous colorimetric and fluorescence method for sensing Tb. The results revealed that the sensitivity of the present assay was about 4 orders of magnitude higher than that of unamplified colorimetric oligonucleotide probe15,53 and was also 1 order of magnitude higher than that of other amplified aptamer-based homogeneous optical assays.22,40,41 Assay reproducibility was also investigated by analyzing a 24.6 pM Tb standard solution nine times. The results showed that the RSD% was found to be 4.3%. Detection Specificity. The developed NESA and DNAzyme amplification based colorimetric aptasensor was also specific. To evaluate this property, we challenged the system with the target Tb and several nontargeted proteins such as IgG, HSA, BSA, FIB, Hb, Mb, zonolysin, GLU, lactic acid, several amino acids, and some metal ions. The results obtained are shown in Figure 6. As can be seen, the specific target Tb led

Figure 7. Results obtained from the testing of buffer solution (control) and different dilution ratios of serum samples without Tb (A) and from the testing of 60-fold diluted serum samples spiked with different concentrations of Tb. The same reaction mixtures without Tb were used as controls (B).

Figure 6. Absorption intensity of the mixture at 418 nm toward Tb, IgG, HSA, BSA, FIB, Hb, Mb, zonolysin, GLU, lactic acid, several amino acids, and some metal ions. The concentrations of Tb was 6.1 nM, and others were 100 nM each.

absorbance values from the diluted serum were comparable to those from buffer solution when the dilution ratio was up to 1:60. Then, the 60-fold dilution of human serum samples were spiked with Tb at three concentrations (6.15, 61.5, and 615 pM Tb) and analyzed. As shown in Figure 7B, comparable responses were found for Tb in both buffer and serum. These results indicated the potentiality of the proposed method for protein detection in real biological samples.

to an increase in the absorbance value, while all nontargeted proteins did not lead to obvious variation of absorption intensity. In addition, two hairpin DNA probes (hairpin DNA-2 and hairpin DNA-3) without aptamer sequences replacing the aptamer hairpin probe respectively for Tb detection was also investigated under the similar conditions. It was found that no change of the absorbance intensity was observed (Supporting Information Figure S3). These results clearly demonstrate the high specificity of the developed NESA and DNAzyme amplification based colorimetric aptasensor. Detection of Thrombin in Human Serum. To demonstrate the feasibility of the approach in complex

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CONCLUSIONS In summary, we have developed a label-free and highly sensitive colorimetric aptasensor for the amplified analysis of biomolecules. As a proof-of-concept, we demonstrate that the developed colorimetric aptasensor can sensitively and selec4428

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tively detect Tb in aqueous solution. This assay has several excellent features. First, the assay does not involve any chemical modification of DNA, which is simple and low-cost. Second, the assay is conducted in aqueous solution, and without requiring troublesome separation procedures. Third, it has high sensitivity and specificity. The detection limit obtained for Tb was 1.5 pM, which is at least 4 orders of magnitude lower over the unamplified colorimetric assay. Finally, this detection system appears to be a universal approach for the detection of target molecules. It could be expanded easily to other targets by simply changing the aptamer sequence of the hairpin probe. Thus, this new methodology can be expected to provide a highly sensitive platform for the amplified analysis of various target molecules.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures for the CD spectra of different samples and optimization of detection conditions and table for performance comparison between homogeneous optical aptasensors. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 773 5856104. Fax: +86 773 5832294. E-mail: [email protected] (S.Z.) and [email protected] (H.L.). Author Contributions §

Y.H. and J.C.: These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundations of China (No. 21175030), the National Basic Research Program of China (No. 2012CB723501), and the Natural Science Foundations of Guangxi Province (No. 2010GXNSFF013001) as well as BAGUI Scholar Program and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), Ministry of Education of China (CMEMR2012-A19).

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