Sensitive Detection of Transcription Factors by Isothermal

Oct 10, 2012 - Here, we develop an isothermal exponential amplification reaction ... of DNA marker-linked antibodies in the case of immuno-PCR and can...
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Sensitive Detection of Transcription Factors by Isothermal Exponential Amplification-Based Colorimetric Assay Yan Zhang, Juan Hu, and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China S Supporting Information *

ABSTRACT: Transcription factors regulate gene expression by binding to specific DNA sequences within the regulatory regions of genes and have become potential targets in clinical diagnosis and drug development. However, traditional approaches for the detection of transcription factors are usually laborious and timeconsuming with a low sensitivity. Here, we develop an isothermal exponential amplification reaction (EXPAR)-based colorimetric assay for simple and sensitive detection of transcription factor NF-κB p50. In this assay, the presence of NF-κB p50 is converted to the reporter oligonucleotides through protein−DNA interaction, exonuclease III digestion, and isothermal exponential amplification. The subsequent sandwich hybridization of the reporter oligonucleotides with the gold nanoparticle (AuNP)-labeled DNA probes generates a red-to-purple color change, allowing the visual detection of NF-κB p50 with the naked eye. Notably, this method converts the detection of transcription factors to the detection of DNA without the requirement of DNA marker-linked antibodies in the case of immuno-PCR and can sensitively measure NF-κB p50 with a detection limit of 3.8 pM, which has improved by as much as 4 orders of magnitude as compared with the conventional AuNPbased colorimetric assay and the label-free luminescence assay and up to 4 orders of magnitude as compared with fluorescence resonance energy transfer (FRET)-based assay as well. Importantly, this method can be used to measure TNF-α-induced endogenous NF-κB p50 in HeLa cell nuclear extracts and might be further applied for the detection of various DNA-binding proteins and aptamer-binding molecules.

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DNA-binding proteins.14 In the presence of DNA-binding protein, two independently labeled DNA fragments, each containing one-half of the binding site of the DNA-binding protein, will bind to the DNA-binding protein, generating distinct fluorescence resonance energy transfer (FRET) signals.14 An alternative FRET-based method for the detection of DNA-binding protein utilizes the doubly labeled oligonucleotides.15,16 Even though the fluorescence-based methods are more convenient than traditional approaches, their complicated procedures in both labeling the DNA probes with fluorescence and designing the proper half-sited DNA molecular beacon limit their broad applications. Therefore, the development of robust methods for simple, cost-effective, and sensitive detection of DNA-binding proteins, especially the transcription factors, is highly desirable. Recently, the colorimetric assay has gained increasing attention because of its excellent selectivity, being easily monitored with the naked eye, and low cost without the requirement of expensive and sophisticated instruments.18−20 Gold nanoparticles (AuNPs) are usually used in the

he DNA-binding proteins play critical roles in the regulation of a variety of essential cellular processes, such as genome replication, gene transcription, cell division, and DNA repair through their binding and interaction with DNA.1−4 The majority of DNA-binding proteins function as the transcription factors to regulate cell development, differentiation, and growth.5−7 Due to their important roles in the pathways and networks of gene expression regulation,8 the transcription factors have become potential targets in clinical diagnosis and drug development.9 Traditional approaches for the detection of DNA-binding proteins include electrophoretic mobility shift assay (EMSA)10 and DNase footprinting assay.11 However, these methods are usually laborious and time-consuming with the involvement of either radioisotopes or fluorescence labels and are not adaptable to high-throughput formats. Alternatively, immunochemical approaches, such as enzyme-linked immunosorbent assay (ELISA)12 and Western blotting assay,13 are very complicated with the requirement of specific antibodies against each DNA-binding protein. Fluorescence-based methods allow the homogeneous assay of DNA-binding proteins in solution and have significant advantages of simplicity, low cost, high sensitivity, and safety without the requirement of radioisotopes.14−17 In these methods, the protein-driven annealing of the two half sites is a typical strategy for the detection of © 2012 American Chemical Society

Received: August 20, 2012 Accepted: October 10, 2012 Published: October 10, 2012 9544

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were mixed at the same molar ratios with the final concentration of 10 μM in the hybridization buffer (50 mM Tris−HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). The mixture was heated at 95 °C for 5 min and then slowly cooled to 25 °C. The purified recombinant NF-κB p50 (rhNF-κB p50) was purchased from Promega (Madison, WI). Exonuclease III (Exo III), Klenow fragment polymerase (3′→5′ exo-, KF polymerase), and Nb.BbvCI nicking endonuclease were obtained from New England Biolabs (Beverly, MA). SYBR Green I was purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). Preparation of DNA-Modified AuNPs. The gold nanoparticles with a diameter of 14 nm were synthesized by the citrate reduction of HAuCl4 following the literature procedures.23 The average diameter of gold nanoparticles was verified by the transmission electron microscope (TEM) (JEM100CXII, Japan). The AuNPs were modified with two different thiol-modified oligonucleotides according to the literature procedures with minor modifications.23 Solutions of 5.2 nmol of probe 1 and 5.2 nmol of probe 2 were added to 2.5 mL of AuNP solution, separately, and incubated at room temperature for 16 h. Then the concentration of phosphate (NaH2PO4/ Na2HPO4) was adjusted to 10 mM by adding 0.1 M concentrated phosphate buffer (pH 7.0), and the concentration of NaCl was adjusted to 0.1 M, followed by standing for 40 h. To remove the excess reagents, the solution was centrifuged at 12 000 rpm for 25 min. After the removal of the supernatant, the red oily precipitate was washed three times with 1 mL of 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl. Then the AuNP-labeled probes were redispersed in the solution of 0.3 M NaCl, 10 mM phosphate buffer (pH 7.0) and stored at 4 °C prior to the use for the detection of transcription factors. Preparation of Cell Extracts. HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin in a humidified chamber containing 5% CO2 at 37 °C. The HeLa cells were cultivated and incubated with or without 20 ng/mL TNF-α (PeproTech, Rocky Hill, NJ) for 30 min, respectively. The nuclear extracts were collected using a nuclear extract kit (ActiveMotif, Carlsbad, CA) according to the manufacturer’s instructions, and the protein concentration was determined by Bradford-based assay. The activity of NF-κB p50 in the TNF-α-treated HeLa cell nuclear extracts was analyzed by EMSA. The reaction solution containing the double-strand DNA (dsDNA) probes and HeLa cell nuclear extracts was incubated in 20 μL of protein binding buffer (10 mM Tris− HCl, pH 7.5, 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, 0.25 mM DTT) at room temperature for 30 min and then subjected to EMSA assay. The SYBR Green I with the final concentration of 1 μg/mL was added into the samples, then the mixture was loaded on a prerun 8% nondenaturing polyacrylamide gel and electrophoresed at 110 V in 1× Tris−borate−EDTA (TBE) buffer. The images of gel electrophoresis were acquired by a Kodak 4000 MM (Kodak, Japan). Protein−DNA Interaction and Exo III Digestion. The purified recombinant NF-κB p50 and dsDNA probes were incubated at room temperature for 30 min in the presence of protein binding buffer containing 10 mM Tris−HCl (pH 7.5), 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mg/mL yeast tRNA, 10% glycerol, 0.25 mM DTT. For the detection of transcription factor NF-κB in the nuclear extracts, the sample was mixed with the DNA probes and incubated at room

colorimetric assay owing to their high extinction coefficients and distance-dependent optical properties.18−20 So far, AuNPbased colorimetric assay has been widely applied for the detection of DNA,21−25 protein,26−30 metal ions,31,32 and small molecules,33−35 as well as the screening of DNA binders.36,37 However, the capability of colorimetric assay in the protein detection is limited by its relatively poor sensitivity due to the lack of suitable approaches for signal amplification. Although the protein targets cannot be chemically duplicated, it is possible to tag the protein targets with the DNA markers which can be subsequently amplified with polymerase chain reaction (PCR), making the protein targets be identified by the standard DNA detection methods (i.e., immmuno-PCR method).38 Nevertheless, the immmuno-PCR method requires special DNA marker-linked antibodies against each protein target. Herein, we develop a simple and sensitive colorimetric assay for the detection of transcription factors by integrating protein−DNA interaction, exonuclease III (Exo III) digestion, and isothermal exponential amplification. The isothermal exponential amplification reaction (EXPAR) as an alternative amplification technique has been used for the detection of DNA39 and miRNA.40,41 In comparison with conventional PCR, the EXPAR provides >106-fold amplification under isothermal conditions within minutes.39 In our method, the integration of protein−DNA interaction, Exo III digestion, and isothermal exponential amplification can convert the detection of transcription factors to the detection of reporter oligonucleotides without the requirement of DNA markerlinked antibodies against each target protein in the case of immuno-PCR assay.38 The subsequent sandwich hybridization of the reporter oligonucleotides with the AuNP-labeled DNA probes generates a red-to-purple color change, allowing the visual detection of NF-κB p50 with the naked eye. As a proof of concept, we demonstrate the capability of the proposed method to quantitatively detect the transcription factor NF-κB p50 with a detection limit of 3.8 pM.

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MATERIALS AND METHODS

Oligonucleotides and Reagents. All oligonucleotides (Table 1) and the deoxynucleotide mixture (dNTPs) were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). To obtain the DNA duplexes, the oligonucleotides Table 1. Sequences of the Oigonucleotidesα note p50s p50anti-s p50s mt p50anti-s mt template reporter DNA probe 1 probe 2

sequence (5′−3′) AGA TGG GAC TTT CCT TGG AAC TAC GAC TCA CTA TAG GGA GAG CAA TTC CAC A TGT GGA ATT GCT CTC CCT ATA GTG AGT CGT AGT TCC AAG GAA AGT CCC ATC T AGA TCT CAC TTT CCT TGG AAC TAC GAC TCA CTA TAG GGA GAG CAA TTC CAC A TGT GGA ATT GCT CTC CCT ATA GTG AGT CGT AGT TCC AAG GAA AGT GAG ATC T ATG GGA CTT TCC TTG GAA CCC TCA GCA GAT GGG ACT TTC CTT GGA ACA−P TGA GGG TTC CAA GGA AAG TCC CAT HS−(T)9AT GGG ACT TTC C TTG GAA CCC TCA (T)9−SH

α

The boldface regions indicate the binding site of NF-κB p50. The underlined bold letters symbolize the mutant bases in the binding site of NF-κB p50. The italic bold letters in the template are the recognition sequence of Nb.BbvCI. 9545

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consists of two reverse complementary oligodeoxynucleotides (ODNs). The binding site of the transcription factor is at the 5′ terminus of a sense ODN strand. Exo III exhibits nonprocessive 3′ to 5′ exodeoxyribonuclease activity which is specific to dsDNA.42 In the presence of transcription factor, Exo III digestion cannot proceed past the binding site,43 and the reverse ODN strand is protected from the digestion and thus preserved. The EXPAR template consists of two copies of the region X separated by the region A. The region X is complementary to the 3′ end of the remaining single-stranded DNA (ssDNA), and the region A is the recognition site for the Nb.BbvCI upon the formation of a dsDNA. In the presence of the EXPAR template, DNA polymerase, endonuclease, and dNTPs, the remaining ssDNA can function as the DNA trigger to initiate the EXPAR to generate a new DNA trigger. This new DNA trigger can bind to another template and initiate a new cycle of polymerization, nicking, and displacement, thus generating a large number of ssDNAs which function as the reporter oligonucleotides. These reporter oligonucleotides can serve as the linker strands to trigger the aggregation of AuNPs and a concomitant color change from red to purple.23 Such color change can be detected immediately with the naked eye and quantified with the absorption spectral measurements. Conversely, in the absence of transcription factors, the naked duplex probes are degraded by Exo III and cannot be amplified by EXPAR. As a result, no reporter oligonucleotide is generated, and no color change is observed. Characterization of the AuNPs and the AuNP−DNA Conjugates. The average diameter of the AuNPs used in this research was about 14 nm as characterized by TEM (Figure 1A). The obtained AuNPs exhibited similar sizes, an almost

temperature for 30 min in the binding buffer containing 10 mM Tris−HCl (pH 7.5), 100 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.25 mM DTT, 2 mM sodium phosphate (pH 7.0), 20 ng/μL HaeIII-cut E. coli DNA, and 25 ng/μL yeast tRNA. Then 2 U/μL Exo III was added to digest the dsDNA probes at 37 °C for 5 min. The digestion reaction was terminated by heating at 70 °C for 20 min. EXPAR Reaction. The EXPAR reaction mixtures were prepared separately as part A and part B. Part A consisted of 2 μL of postdigestion mixture, 0.05 μM template, 250 μM deoxynucleotide triphosphates (dNTPs), 1× NEB buffer 2 (10 mM Tris−HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9). Part B consisted of 0.25 U/μL Nb.BbvCI nicking endonuclease and 0.05 U/μL KF polymerase. The reactions were carried out in a volume of 20 μL. Part A was heated at 95 °C for 3 min, followed by incubation at 40 °C for 5 min. Then parts A and B were added immediately, and the reactions were processed at 40 °C for 40 min. The EXPAR products were kept at 4 °C for subsequent analysis by nondenaturing polyacrylamide gel electrophoresis (PAGE). AuNP-Based Colorimetric Assay. The sandwich hybridization was carried out by mixing 10 μL of EXPAR products with 30 μL of solutions containing a 1:1 ratio of AuNP-labeled DNA probe 1 and AuNP-labeled DNA probe 2. The solution was diluted to 150 μL with a buffer containing 0.3 M NaCl and 10 mM phosphate buffer (pH 7.0). The UV−vis absorption spectra were recorded on a Perkin Elmer Lambda 25 UV−vis spectrophotometer (Perkin Elmer, Waltham, MA, U.S.A.).

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RESULTS AND DISCUSSION

Principle of EXPAR-Based Colorimetric Assay for the Detection of Transcription Factors. The principle of EXPAR-based colorimetric assay for the detection of transcription factors is presented in Scheme 1. The dsDNA probe Scheme 1. Schematic Illustration of EXPAR-Based Colorimetric Assay for the Detection of Transcription Factors

Figure 1. (A) TEM image of the AuNPs. (B) Normalized UV−vis spectra of the AuNPs (a) and the DNA-modified AuNPs (b).

round shape, and good monodispersity. As individual particles, the AuNPs had a maximum absorbance at a wavelength of 520 nm (Figure 1B, curve a). The DNA-modified AuNPs displayed a modest shift in the surface plasmon band from 520 to 525 nm (Figure 1B, curve b) as reported in the literature.23 In addition, the well-functionalized AuNPs maintained the red color as the plain AuNPs and had high stability in the solution containing high salt concentration (0.3 M NaCl). Detection of NF-κB p50 with EXPAR-Based Colorimetric Assay. To demonstrate the capability of EXPAR-based colorimetric assay for the detection of transcription factors, NFκB p50 was chosen as a model. NF-κB p50 can bind to the consensus DNA of GGG ACT TTC C in a sequence-specific manner.44 The binding of NF-κB p50 to the probe can protect the reverse DNA strand from being degraded by Exo III and subsequently produces a large number of reporter oligonucleotides by EXPAR. To obtain the high amplification efficiency of EXPAR, the reaction temperature was optimized, and 40 °C 9546

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was selected as the optimum reaction temperature (see the Supporting Information, Figure S-1). The amplification products were confirmed by 14% PAGE, and a well-defined band of the reporter oligonucleotides (24 nt) was observed in the presence of NF-κB p50 (Figure 2A, lane 1). In contrast, no

Figure 2. (A) Nondenaturing PAGE analysis of EXPAR amplification products: lane 1, in the presence of 8 nM NF-κB probes and 8 nM NFκB p50; lane 2, in the presence of 8 nM NF-κB probes without NF-κB p50; lane 3, the reporter oligonucleotide (24 nt); lane M, the DNA ladder marker. (B) Absorption spectra obtained from the hybridization of EXPAR amplification products with the AuNP-labeled DNA probes in the presence of 2 nM NF-κB p50 (red line) and in the absence of NF-κB p50 (black line). The concentration of NF-κB probes is 2 nM. The inset shows the corresponding color changes.

distinguishable band was observed in the negative control without NF-κB p50 (Figure 2A, lane 2). In the presence of amplification products, the hybridization of AuNP-labeled DNA probes with the reporter oligonucleotide created a DNA-guided self-assembly of AuNPs. Both a red-to-purple color change (inset in Figure 2B) and a significant absorption spectral shift (Figure 2B, red line) were observed in the presence of NF-κB p50. In contrast, neither apparent color change (inset in Figure 2B) nor absorption spectral shift (Figure 2B, black line) was observed in the absence of NF-κB p50. These results clearly demonstrated that the proposed method could be used successfully for the detection of transcription factors. Specificity of EXPAR-Based Colorimetric Assay. The specificity of the proposed method was evaluated with two control experiments that involved (1) the nonspecific sequence of NF-κB p50 and (2) an irrelevant protein (bovine serum albumin, BSA), respectively. A series of sequences that possessed various affinities for NF-κB p50 had been identified.16,45 To test the specificity of the proposed method, we first changed GGG to CTC in the sense strand of specific binding site of NF-κB p50 (Table 1). Equal concentrations of specific and nonspecific sequence of NF-κB p50 were incubated with NF-κB p50. After digestion and amplification, the colorimetric assay displayed both an obvious red-to-purple color change (inset in Figure 3A) and an absorption spectral shift (Figure 3A, curve a) in the presence of specific sequence. In contrast, there was neither color change (inset in Figure 3A) nor absorption spectral shift (Figure 3A, curve b) observed in the presence of nonspecific sequence as compared with the control without NF-κB p50 even in the presence of specific sequence (inset in Figure 3A and Figure 3A, curve c). These results indicated the high specificity of the proposed method for the detection of transcription factors. In the second control experiment, the selectivity of the proposed method was investigated in the presence of BSA which showed no binding

Figure 3. (A) Absorption spectra of AuNPs in response to 2 nM NFκB p50 in the presence of 2 nM specific probes (a), 2 nM nonspecific probes (b), and 2 nM specific probes without NF-κB p50 (c). (B) Absorption spectra of AuNPs in response to 2 nM NF-κB p50 (green line), 2 nM BSA (red line), and the control without NF-κB p50 (black line) in the presence of 2 nM NF-κB probes. The inset shows the corresponding color changes.

to the specific sequence of NF-κB p50. Neither color change (inset in Figure 3B) nor spectral shift (Figure 3B, red line) was observed in the presence of BSA. In the contrary, both an obvious color change (inset in Figure 3B) and a remarkable spectral shift (Figure 3B, green line) were observed in the presence of NF-κB p50. These results indicted the high selectivity of the proposed method for the detection of transcription factors. Improved Sensitivity for NF-κB p50 Detection. To demonstrate the improved sensitivity of the proposed method, NF-κB p50 with different concentrations was measured. As shown in Figure 4A, an obvious color change from red to purple was observed with the increase in the concentration of NF-κB p50. Meanwhile, in the UV−vis spectra, the absorbance at 525 nm decreased, whereas the absorbance at 700 nm increased with the increasing concentration of NF-κB p50 (Figure 4B). In this research, the absorbance ratio at these two wavelengths (A700/A525) was used for quantitative analysis with a high ratio in the case of purple-colored aggregates and a low ratio in the case of red-colored dispersed particles. As shown in Figure 4C, the ratio of A700/A525 increased monotonically with the increasing concentration of NF-κB p50, indicating that the concentration of NF-κB p50 was proportional to the remaining copy number of ssDNA.46 In addition, the absorption ratio of A700/A525 exhibited a linear correlation to the logarithm (log) of the concentration of NFκB p50 over a range of 3 orders of magnitude from 5 pM to 2 9547

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the proposed method was comparable with that of real-time PCR,47 but the proposed method did not require the expensive doubly labeled TaqMan probes and high-precision thermal cycling in the case of real-time PCR, thus significantly reducing the experimental cost and complexity. Real Sample Analysis. To further demonstrate the capability of the proposed method for real sample analysis, the endogenous NF-κB p50 in HeLa cell nuclear extracts was measured. Because the endogenous nuclease in the cell nuclear extracts may influence the Exo III activity,48 the protein binding buffer containing sodium phosphate, poly(dI-dC), HaeIII-cut E. coli DNA, and yeast tRNA was used in this research to efficiently suppress the activity of endogenous nuclease.48,49 TNF-α was used to stimulate the NF-κB activation in HeLa cell nuclear extracts.50 The activity of NF-κB p50 in the nuclear extracts was analyzed by EMSA. A distinct band of NF-κB p50−dsDNA complex was observed in the TNF-α-treated nuclear extracts (Figure 5A, lane 3). In contrast, a relatively low

Figure 5. (A) Detection of endogenous NF-κB activity by EMSA in HeLa cell nuclear extracts: lane 1, in the presence of 10 μg of untreated nuclear extracts and 4 pmol NF-κB probes; lane 2, in the presence of 10 μg of TNF-α-treated nuclear extracts without NF-κB probes; line 3, in the presence of 10 μg of TNF-α-treated nuclear extracts and 4 pmol NF-κB probes. (B) Absorption spectra of the AuNPs in response to NF-κB p50 in the HeLa cell nuclear extracts in the presence of 2 nM NF-κB probes: (a) TNF-α-treated nuclear extracts (25 ng/μL); (b) untreated nuclear extracts (25 ng/μL); (c) the control without the nuclear extracts. The inset shows the corresponding color changes.

Figure 4. (A) Photograph and (B) absorption spectra of the AuNPs in response to different concentrations of NF-κB p50 in the presence of 2 nM NF-κB probes. (C) Variance of absorption ratio of A700/A525 as a function of the concentration of NF-κB p50. Inset: the absorption ratio of A700/A525 is a log−linear correlation with the concentration of NF-κB p50 in the range from 5 to 2000 pM. Error bars show the standard deviation of three experiments.

EMSA signal was observed in the untreated nuclear extracts (Figure 5A, lane 1). In accordance with the results of EMSA, an obvious color change (inset in Figure 5B) and a large absorbance variance (Figure 5B, curve a) were observed in the TNF-α-treated HeLa cell nuclear extracts, while the untreated cell nuclear extracts displayed negligible color change (inset in Figure 5B) and small absorbance variance (Figure 5B, curve b) as compared with the control without the nuclear extracts (inset in Figure 5B and Figure 5B, curve c). These results indicated the capability of the proposed method for the detection of NF-κB p50 in real samples.

nM (inset in Figure 4C). The correlation equation was A = −0.0316 + 0.2731 log10 C (R2 = 0.9977), where A was the absorption ratio of A700/A525 and C was the concentration of NF-κB p50 (picomolar). The detection limit of 3.8 pM was obtained by evaluating the average response of the negative control plus 3 times standard deviation. Notably, the sensitivity of the proposed method had improved by as much as 4 orders of magnitude as compared with the previously reported AuNPbased colorimetric assay29 and the label-free luminescence assay45 and up to 4 orders of magnitude as compared with FRET-based assay as well.14,15 Such significant improvement in the detection sensitivity might be attributed to (1) the high specificity of NF-κB probes to NF-κB p50, (2) the excellent protection offered by DNA−protein binding to prevent the dsDNA from the digestion by ExoIII, and (3) the high amplification efficiency of EXPAR. In addition, the sensitivity of

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CONCLUSION In summary, we have developed an EXPAR-based colorimetric assay for simple and sensitive detection of transcription factors. This method can convert the detection of transcription factor to the detection of DNA without the requirement of DNA marker-linked antibodies in the case of immuno-PCR.38 Due to the excellent specificity of the DNA probes to the transcription factors and the high amplification efficiency of EXPAR, this method can sensitively measure NF-κB p50 with a detection limit of 3.8 pM, which has improved by as much as 4 orders of 9548

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magnitude as compared with the conventional AuNP-based colorimetric assay.29 In comparison with fluorescence-based assay,14−17 this method has significant advantages of low cost without the requirement of fluorescent-labeled nucleotides and direct visualization with the naked eye without the requirement of expensive and sophisticated instruments. In addition, this method can be used to measure TNF-α-induced endogenous NF-κB p50 in HeLa cell nuclear extracts and might be further extended to detect a variety of DNA-binding proteins and aptamer-binding molecules in the biomedical research and early clinical diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure S-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant No. 21075129), the Guangdong Innovation Research Team Fund for Low-Cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant No. JC201005270327A), the Fund for Shenzhen Engineering Laboratory of Single-Molecule Detection and Instrument Development (Grant No. (2012) 433), and the Award for the Hundred Talent Program of the Chinese Academy of Science.

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REFERENCES

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dx.doi.org/10.1021/ac3024087 | Anal. Chem. 2012, 84, 9544−9549