General Colorimetric Detection of Proteins and Small Molecules

May 29, 2012 - However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an apta...
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General Colorimetric Detection of Proteins and Small Molecules Based on Cyclic Enzymatic Signal Amplification and Hairpin Aptamer Probe Juan Li, Hua-E. Fu, Ling-Jie Wu, Ai-Xian Zheng, Guo-Nan Chen, and Huang-Hao Yang* The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002 (P.R. China) S Supporting Information *

ABSTRACT: In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV−vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.

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appropriate dye for a designated aptamer. Recently, Soh and coworkers have developed an aptamer-functionalized polydiacetylene liposome sensor for colorimetric detection of thrombin while maintaining low detection sensitivity.16 In colorimetric methods, gold nanoparticles (AuNPs) have emerged as the most important colorimetric reporters because of their high extinction coefficients and strong size-dependent SPR properties.19 The color of the AuNPs solution can change from red to purple, in response to the SPR absorption of dispersed and aggregated nanoparticles. Such obvious color change is sensitive, thus it is considered ideal in designing colorimetric sensing platforms. AuNPs-based colorimetric methods have been successfully employed for the analysis of several analytes, including small molecules,20,21 metal ions,22 DNA,23 and proteins.24−26 Albeit substantial progress was accomplished, one of the common limitations of AuNPs-based methods lies in the relatively poor sensitivity, which is mainly caused by the lack of amplification of the detection signal.27 For example, Lu and coworkers have developed a three-component sandwich colori-

ptamers are single-stranded nucleic acids isolated from random-sequence DNA or RNA libraries by an in vitro selection process termed the systematic evolution of ligands by exponential enrichment (SELEX).1 They possess high recognition ability toward specific molecular targets ranging from small inorganic and organic substances to even a protein or cell.2−4 Compared with other recognition elements, such as antibodies, aptamers have multifarious advantages, such as simple synthesis, good stability, design flexibility and wide applicability, making them suitable candidates for biological application.5−8 Since their first discovery in the 1990s, aptamers have been greatly focused in the area of biosensors, bringing about a new branch as aptasensors.9 The key issue in the development of aptasensors is how to convert target recognition and binding into a measurable signal. Up until now, many methods have been used for this purpose, including electrochemistry,10,11 fluorescence,12,13 colorimetry,14−16 surface plasmon resonance (SPR),17 atomic force microscope (AFM),18 and so on. Among various methods, simple colorimetric methods have attracted significant interest due to its rapidness, simplicity, and no need to use expensive analytical instruments. For example, organic-dye replacement was employed to design a colorimetric cocaine sensor.14,15 However, it is hard to find an © 2012 American Chemical Society

Received: March 2, 2012 Accepted: May 29, 2012 Published: May 29, 2012 5309

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cm resistivity) was used in all runs. Other chemicals were all of analytical grade and used as received. The morphology of the AuNPs was observed by a Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) at 200 kV. UV−vis absorption spectra were recorded in 1 cm path length quartz cuvettes on a Perkin-Elmer Lambda 750 UV−vis spectrometer. Preparation of AuNPs and DNA-Modified AuNPs. AuNPs with an average diameter of 13 nm were prepared according to a previously described protocol.23 In brief, an aqueous solution of HAuCl4 (1 mM, 50 mL) was brought to reflux while stirring. Then 5 mL of trisodium citrate solution (38.8 mM) was added quickly, resulting in a change in solution color from pale yellow to deep red. The solution was heated under reflux for 10 min and then allowed to cool to room temperature. The size of AuNPs was verified by TEM, and their concentration was estimated by UV−vis spectroscopy. The DNA-modified AuNPs were prepared as follows. The thiolmodified DNA 1 and DNA 2 were first activated by 2 equiv of TCEP for 1 h, respectively. Then, TCEP-activated DNA 1 and DNA 2 were incubated with AuNPs and were mixed at room temperature for 16 h, and then the mixture was aged in salt and brought to a final concentration of 0.1 M NaCl through a stepwise process. This mixed solution was further incubated for 24 h at room temperature. To remove excess reagents, the solution was centrifuged for 30 min at 12 000 rpm. The precipitate was washed three times with a TA buffer (20 mM Tris-acetate, pH7.9), with repetitive centrifugation and dispersion, which was finally dispersed in the TA buffer and stored at 4 °C until use. Preparation of ATP Extracts from Cancer Cells. The HeLa cell was used in this study. The cell density was determined by a hemocytometer prior to each experiment. Then, a suspension of 2.2 × 106 cells dispersed in RPMI-1640 cell buffer and was centrifuged at 1000 rpm for 5 min, then washed with phosphate-buffered saline five times. Finally, the cells were disrupted by sonication for 20 min at 0 °C. To remove the homogenate of cell debris, the lysate was centrifuged at 18 000 rpm for 20 min at 4 °C. Then, the cell lysate was treated with deproteination by filtration using cutoff membranes. For comparison, ATP levels were assayed with a HPLC method as previously reported.36 Fabrication of Colorimetric Method Based on Cyclic Enzymatic Signal Amplification (CESA). The detailed procedure of the fabrication of CESA-based method was as follows. First, the thrombin hairpin aptamer probe or ATP hairpin aptamer probe was mixed with different concentrations of thrombin or ATP at 37 °C for 30 min to form the aptamer probe-target complex, respectively. Second, the linker DNA and the different concentrations of NEase were added to the solution at 37 °C for the required reaction time. Then, the obtained mixtures were heated at 80 °C for 20 min to terminate the reaction. Third, the mixtures were finally added to the solution containing DNA 1 functionalized AuNPs and DNA 2 functionalized AuNPs. The resulting samples were photographed and tested with a UV−vis spectrometer.

metric assay for adenosine based on AuNPs with a detection limit of 300 μM.20 Recently, Dong and co-workers have developed a AuNPs-based colorimetric assay for thrombin which provides a detection limit of 83 nM.24 To address this drawback, the development of amplification strategies for AuNPs-based methods is remarkably important. Up until now, several signal amplification strategies have been reported.28−35 Nevertheless, there are only a few amplified strategies available for development of colorimetric methods based on AuNPs.33−35 Recently, Li and co-workers proposed a smart nicking endonuclease-based aptasensor (NEBAS) for the amplified detection of potassium.35 However, this NEBAS can only be applied to the aptamer with the sequence containing the recognition site of the nicking endonuclease. On the other hand, only a very limited numbers of nicking endonuclease is commercially available now. So, although this NEBAS is sensitive and simple, the generality remains a big problem. To the best of our knowledge, there are few universal amplified strategies available for development of highly sensitive AuNPs-based aptasensors. Herein, taking advantage of cyclic enzymatic signal amplification (CESA) and a hairpin aptamer probe, we designed a general colorimetric method for amplified detection of proteins and small molecules.

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EXPERIMENTAL SECTION Materials and Apparatus. Oligonucleotides used in this study were synthesized by Shanghai Sangon Biotechnology Co., which were purified by HPLC and confirmed by mass spectrometry. Table 1 shows the sequences of the used Table 1. Sequences of the Used Oligonucleotides (in the 5′ to 3′ Direction)a type

sequence

Thr-H1

5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CTG CAA AAA AAT CCT CAG CAG TCA CCC-3′ 5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CTG CAA AAA AAT CCT CAG CAG TCA CCC C −3′ 5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CTG CAA AAA AAT CCT CAG CAG TCA CCC CC-3′ 5′-ACC TGG GGG AGT ATT GCG GAG GAA GGT GCA AAA AAA TCC TCA GCA CCT TC-3′ 5′-ACC TGG GGG AGT ATT GCG GAG GAA GGT GCA AAA AAA TCC TCA GCA CCT TCC −3′ 5′-ACC TGG GGG AGT ATT GCG GAG GAA GGT GCA AAA AAA TCC TCA GCA CCT TCC C-3′ 5′- GCA AAC AAC TGC↓TGA GGA TAA ACG-3′

Thr-H2 Thr-H3 ATPH1 ATPH2 ATPH3 linker DNA DNA 1 DNA 2

5′-SH-(CH2)6-CGT TTA TCC TCA-3′ 5′-GCA GTT GTT TGC-(CH2)6-SH-3′

a

The italic bold letters of Thr-H1, Thr-H2, and Thr-H3 are the sequence of thrombin aptamer. The italic bold letters of ATP-H1, ATP-H2, and ATP-H3 are the sequence of the ATP aptamer. The underlined bold letters of linker DNA and the entire hairpin aptamer probes are the recognition sequence of NEase, and the arrow indicates the nicking position.

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oligonucleotides. Adenosine triphosphate (ATP) and all proteins were purchased from Sigma-Aldrich Chemical Co. NEase (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. Chloroauric acid (HAuCl4), tris(2-carboxyethyl)phosphine (TCEP), and citric acid were purchased from Aldrich. Ultrapure water obtained from a Millipore water purification system (18 MΩ

RESULTS AND DISCUSSION Design of Cyclic Enzymatic Signal Amplification (CESA)-Based Colorimetric Method. The principle of CESA-based method is illustrated in Scheme 1. The CESA is accomplished by nicking endonuclease-strand scission cycle. Our detection system consists of a hairpin aptamer probe, linker 5310

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Scheme 1. Schematic Illustration of General Colorimetric Method Based on CESA

Figure 1. TEM images of (A) the as-prepared AuNPs, (B) the DNA-modified AuNPs solution after adding the mixture of linker DNA, hairpin aptamer probe and NEase, (C) the DNA-modified AuNPs solution after adding the mixture of linker DNA, hairpin aptamer probe, NEase, and 10 nM thrombin.

the strand-scission cycle, two sets of DNA-modified AuNPs are added to the solution to quantify the amount of linker DNA left. The cleaved fragments of linker DNA can no longer be used to assemble the DNA-modified AuNPs, thus a red color of separated AuNPs could be observed. If only a fraction of linker DNA is cleaved, the system will be made of a mixture of aggregated and separated AuNPs, which gives a purple color. In this way, by transferring the quantitative information of target to that of a linker DNA, the method can be transferred to DNA detection, which is subsequently detected by the colorimetric assay based on AuNPs. Viability of the Design for Detection of Protein. To demonstrate the utility of our design, we employed thrombin as a model target protein. Thrombin has played significant roles in the coagulation cascade, thrombosis, and hemostasis and relates to a multitude of diseases.41 Therefore, sensitive detection of thrombin is critical to many areas of biomedical research and diagnostics. Transmission electron microscopy (TEM) and UV−visible spectroscopy were performed to investigate the viability of our design. As shown in Figure 1A, the diameter of the as-prepared AuNPs is about 13 nm. A red color of separated AuNPs is observed. When an appropriate amount of linker DNA was added in the AuNPs solution, the solution color changed from red to blue and the aggregation of AuNPs could be observed by TEM. Furthermore, after the addition of hairpin aptamer probe and NEase into the above AuNPs solution, the solution color did not display differences (Figure 1B). This result demonstrated the hairpin aptamer probe and linker DNA can stably coexist in solution. The linker DNA can effectively assemble the two sets of DNA-modified AuNPs through DNA hybridization, inducing aggregation of AuNPs. In contrast, in the presence of thrombin, the TEM image of the AuNPs shows a majority of dispersed particles (Figure 1C). These results proved that target could interact with the hairpin aptamer probe to form a probe-target complex, which can hybridize to the linker DNA to form a full

DNA, two sets of DNA-modified AuNPs, and NEase. Numerous hairpin aptamer structures were used to fabricate biosensors.37−39 NEase is a special family of restriction endonucleases, which can recognize a specific sequence known as a restriction site along a double-strand DNA and only cleave one strand of it, leaving a nick in the DNA.40 The NEase used here is Nb.BbvCI, which recognizes a simple asymmetric sequence, 5′-GCTGAGG3′. It must be mentioned that it is feasible to select other NEases to complete the design since there is no requirement of the NEase recognition site in the aptamer sequence. The hairpin aptamer probe contains three domains termed as I, II, and III according to their different functions. The region I is the sequence of aptamer (colored blue), which is partially caged in the duplex structure of the stem by hybridization with region III. While the region II is a single-stranded loop, which is complementary to the linker DNA and simultaneously contains a NEase recognition sequence (colored green). In the absence of target, the hairpin aptamer probe and linker DNA can stably coexist in solution because of the low hybridization efficiency. After the addition of two sets of DNAmodified AuNPs with sequences complementary to the two ends of the linker DNA, respectively, the linker DNA can assemble the DNA-modified AuNPs, inducing the aggregation of AuNPs. Also, a color change from red to blue can be observed. However, in the presence of target, the hairpin structure of the aptamer probe is opened upon interaction of region I with the target to form the probe-target complex. Thereby, region II of the probe can hybridize to the linker DNA. Upon formation of the duplex, the NEase can recognize the specific nucleotide sequence and cleave the linker DNA into two fragments. After nicking, the duplex becomes unstable and the cleaved linker DNA fragments will dissociate from the probe-target complex. Then, the probetarget complex can hybridize with another intact linker DNA to form a new substrate for NEase and the cycle starts anew. Through such strand-scission cycle, only one target can trigger cleavage of a large quantity of linker DNA. Upon completion of 5311

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A610 under conditions with or without 5 nM thrombin, the ThrH2 showed a larger signal-to-noise ratio than Thr-H1 and ThrH3. The Thr-H1 has relatively less stable structure, which is relatively easier to be opened directly by linker DNA to form duplex DNA. Such duplex DNA could be recognized and cleaved by the NEase, resulting in high background signal and low sensitivity. The Thr-H2 probe was the most effective probe in the group and had a significant signal enhancement. The Thr-H3 has the most stable structure and therefore a rather low ability to interact with target, resulting in the decrease of signal enhancement. Thus, the Thr-H2 probe was chosen as the optimum probe in the experiments. The concentration of NEase also plays an important role in the sensing process. To investigate the effect of the concentration of NEase, Thr-H2, linker DNA, and thrombin were mixed with different concentrations of NEase ranging from 0 U to 20 U (Figure 3A). The ratio of absorbance intensities (A520/A610) increased with an increasing concentration of NEase, implying enhanced cleavage of the linker DNA. Meanwhile, it can be observed that the background signal also increased with

recognition site for the NEase. Then the NEase can cleave the linker DNA into two fragments. The AuNPs solution remains red in color. To further confirm that the above changes of the optical properties of AuNPs were due to cleavage of the linker DNA, the assay for thrombin with or without NEase was carried out. Figure 2 depicts the absorption spectra of DNA-modified AuNPs under

Figure 2. Absorption spectra of DNA-modified AuNPs for detection of thrombin (a) without NEase, (b) with NEase (5 U), and (c) with thermally inactivated NEase (5 U). The reaction system contained thrombin hairpin aptamer probe (50 nM), linker DNA (30 nM), and thrombin (10 nM).

different conditions. In the absence of NEase, a broad absorption for the AuNPs aggregates appeared at 550−700 nm (curve a). The results demonstrated that, in the absence of the NEase, thrombin with low concentration (10 nM) did not interfere with the hybridization between the linker DNA and the DNAmodified AuNPs. In contrast, in the presence of NEase, a narrow absorption peak at 520 nm for dispersed AuNPs was observed (curve b). As a control, NEase was inactivated by heating at 80 °C for 20 min and then introduced in the system. In this case, the aggregated AuNPs were also obtained (curve c) and the corresponding absorption spectrum showed no significant difference from that of the system without NEase (curve a). Comparing curve b with curve c, it is clear that NEase can work in the system and effectively degrade the linker DNA hybridizing with the probe-target complex. The UV−visible spectroscopy served as complementary evidence along with TEM results to clearly support our concept for detection of protein based on CESA. Optimization of Experimental Conditions for Protein Detection. In this study, we used the ratio of absorbance intensities at 520 and 610 nm (A520/A610) to assess the degree of aggregation, which is reported to be more accurate for analysis. The ratio is also associated with the color of the solution, with a high ratio corresponding to a red solution and a low ratio corresponding to a purple one. In order to achieve the best sensing performance, the stability of the hairpin aptamer probe, the concentrations of NEase, and nicking time were investigated. At first, to optimize the hairpin aptamer probe, we synthesized three different hairpin aptamer probes (Thr-H1, Thr-H2, and Thr-H3) that included 10, 11, and 12 base pairs in the stem region, respectively (as shown in Table 1). As shown in Figure S1 in the Supporting Information, according to the change of A520/

Figure 3. (A) The absorption ratio was plotted as a function of the NEase concentration in the absence (black line) or in the presence (red line) of thrombin, respectively. The system contained Thr-H2 (50 nM), linker DNA (30 nM), and thrombin (5 nM). (B) The absorption ratio was plotted as a function of the nicking time, in the absence (black line) or in the presence (red line) of thrombin, respectively. The system contained Thr-H2 (50 nM), linker DNA (30 nM), thrombin (5 nM), and NEase (5 U). 5312

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thrombin. As shown in Figure 4A, in the absence of thrombin, the linker DNA can hardly be cleaved by NEase and consequently can assemble two sets of DNA-modified AuNPs. Thus, a blue color of aggregated AuNPs was observed. As expected, in the presence of thrombin, the linker DNA was cleaved by NEase. Also the cleaved linker DNA was not able to assemble DNAmodified AuNPs. Furthermore, the rate of the cleavage reaction can be modulated by the concentration of thrombin, and the amount of cleaved linker DNA at a set time is dependent on the concentration of thrombin. Then, different ratios of the cleaved DNA to noncleaved linker DNA will result in different extents of AuNPs aggregation and thus different colors from blue to red. Meanwhile, in the UV−vis spectra, the absorbance at 520 nm increased, whereas that at 610 nm decreased (Figure 4B). Also the ratio of UV−vis absorption at 520 and 610 nm (A520/A610) continued to increase with the increase of thrombin concentration until a plateau was reached (Figure 4C). In addition, the absorption ratio was linear to the concentration of thrombin in the range from 50 pM to 5 nM (Figure 4C, inset). This assay allowed for the detection of thrombin at concentration as low as 50 pM by the naked eye (Figure 4A). The detection limit (defined as 3σ, where σ is the standard deviation of the blank) was 20 pM by UV−vis spectroscopy. This high sensitivity is due to the large signal amplification upon the addition of NEase. The sensitivity of this proposed method is about 3 orders of magnitude higher than that of the previously reported colorimetric detection of thrombin.24 The selectivity of the proposed method described herein was determined by examining the optical responses of system toward human IgG, hemoglobin, human serum albumin (HSA), and bovine serum albumin (BSA). The system showed red color in the presence of thrombin. Nevertheless, in the presence of other four kinds of nonspecific proteins, the color of the system turned blue under the same conditions (Figure 5). The above results

increasing the concentration of NEase. The results indicated that the optimum concentration of NEase was 5 U due to its best signal-to-noise level. Kinetic studies were then performed to study the nicking process by monitoring the A520/A610 as a function of time (Figure 3B). We recorded the ratio under conditions with or without thrombin. In the presence of thrombin, the ratio elevated at a fast rate in the initial stage and was followed by a slow increase after 60 min (red line). However, the background maintained its increased rate even after 60 min (black line). Therefore, for the best signal-to-noise level, 60 min was selected for the nicking reaction in this work. CESA-Based Colorimetric Detection of Protein. By employing our design, we demonstrated that this colorimetric method has high sensitivity. Figure 4 shows the color and absorption of system in the presence of various concentrations of

Figure 5. (A) Photograph and (B) absorption spectra of system toward thrombin (5 nM) and different nonspecific proteins (5 nM). a = human IgG, b = hemoglobin, c = HSA, d = BSA, and e = thrombin. The details are provided in the Experimental Section. Figure 4. (A) Photograph and (B) absorption spectra of the system after incubation with different concentrations of thrombin (a−h, 0, 0.05, 0.1, 1, 2, 5, 10, and 20 nM, respectively). (C) The absorption ratio (A520/ A610) was plotted as a function of the concentration of thrombin. Inset: magnification of the plot in the range 0.05−5 nM. The data shown here represent the means and standard deviations of three independent experiments.

clearly demonstrated that the developed colorimetric method held potentials for quantitative assay of the target proteins with high sensitivity and selectivity. Viability of the Design for Detection of Small Molecules. To explore the generalizability of our design, we applied this amplified colorimetric method to detect small 5313

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triphosphate (GTP), and uridine triphosphate (UTP) were employed for the control experiments. The system showed red color in the presence of ATP. Nevertheless, in the presence of three analogues, the color of the system turned blue under the same conditions (Figure 7). Such obvious difference in color

molecules. We employed ATP as the model target analyte. ATP plays critical roles in the regulation and integration of cellular metabolism and biochemical pathways in cell physiology, which has also been used as an indicator for cell viability and cell injury.42 Therefore, determination of ATP is essential in biochemical study as well as clinical diagnosis. Similarly, in order to achieve the best sensing performance, the stability of the ATP hairpin aptamer probe was optimized. We synthesized three probes (named as ATP-H1, ATP-H2, and ATP-H3), which include 8, 9, and 10 base pairs in the stem region, respectively (as shown in Table 1). Experimental results showed that the ATPH2 provide a maximum signal-to-noise level for the sensing system (Supporting Information, Figure S2). The concentration of NEase and the nicking time were also optimized (Supporting Information, Figures S3 and S4). Under the optimization conditions, the color and absorption of DNA-modified AuNPs in the presence of the ATP-H2, linker DNA, NEase, and various concentrations of ATP are shown in Figure 6. The color of system was correlated to the

Figure 7. (A) Photograph and (B) absorption spectra of system toward ATP (10 μM), CTP (10 μM), UTP (10 μM), and GTP (10 μM). The details are provided in the Experimental Section.

suggested that the proposed method in this work is of considerable selectivity. Since high sensitivity and selectivity, we considered applying the proposed method to analyze ATP in complicated biological samples. In this case, analysis of cellular ATP from HeLa cell lysate was implemented. The results of the determination were listed in Table 2. The detected concentration Table 2. Comparisons of CESA-Based Colorimetric Method with the HPLC Method for the Detection of ATP in the HeLa Lysate samplea

this method (μM)

RSD (%, n = 3)

HPLC (μM)

RSD (%, n = 3)

1 2 3

3.69 3.94 3.83

3.9 3.1 4.2

3.86 3.78 3.61

3.2 3.7 4.1

a

Each sample was analyzed in triplicate, and the results are the average values.

Figure 6. (A) Photograph of system after incubation with different concentrations of ATP in the presence of 10 U NEase. (B) The absorption ratio (A520/A610) was plotted as a function of the concentration of ATP. Inset: magnification of the plot in the range 0.05−10 μM. The data shown here represent the means and standard deviations of three independent experiments.

of ATP in the HeLa lysate was about 3.82 μM, which was in good agreement with that obtained using the HPLC method. These results demonstrated that the developed method could be used to monitor the content of ATP in cell extracts without the interference of other substances in the cell.

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concentrations of ATP (Figure 6A). Meanwhile, the ratio of UV−vis absorption at 520 and 610 nm (A520/A610) was linear to the concentration of ATP in the range from 50 nM to 10 μM (Figure 6B). These results demonstrated that the proposed method can be used for highly sensitive detection of ATP by the naked eye. The detection limit (defined as 3σ, where σ is the standard deviation of the blank) was 25 nM by UV−vis spectroscopy and 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of colorimetric detection without NEase (Supporting Information, Figure S5). Application of Method to Detect ATP in Real Samples. We further examined the selectivity of the method for ATP. ATP analogues such as cytidine triphosphate (CTP), guanosine

CONCLUSIONS In conclusion, we have demonstrated a general colorimetric method based on gold nanoparticles, cyclic enzymatic signal amplification, and a hairpin aptamer probe for highly efficient detection of proteins and small molecules. Using this colorimetric method, we detected the human thrombin with a detection limit of 50 pM and ATP with a detection limit of 100 nM by the naked eye, respectively. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. It allows simple and direct visualization detection by using the naked eye, which does not 5314

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require any power or complicated instrumentation. In addition, this method is general since there is no requirement of the nicking endonuclease recognition site in the aptamer sequence. Moreover, the proposed method is capable of detecting target in complicated biological samples such as cell lysate. Therefore, this simple and highly sensitive colorimetric method should find applications in a diverse range of areas, such as medical diagnostics, environmental monitoring, and the electronic industry.

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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: hhyang@fio.org.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial from the National Basic Research Program of China (Grant No. 2010CB732403), the National Natural Science Foundation of China (Grant No. 21125524, Grant No. 20975023), the Program for New Century Excellent Talents in University of China (Grant 09-0014), the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1116), and the National Science Foundation of Fujian Province (Grant 2010J06003).

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dx.doi.org/10.1021/ac3006186 | Anal. Chem. 2012, 84, 5309−5315