Label-Free Electrochemical Detection of the p53 Core Domain Protein

Anal. Chem. 2009, 81, 4770–4777

Label-Free Electrochemical Detection of the p53 Core Domain Protein on Its Antibody Immobilized Electrode Jongchan Yeo,†,‡ Jin-Young Park,†,‡ Won Jin Bae,§ Yoon Suk Lee,† Byeang Hyean Kim,† Yunje Cho,§ and Su-Moon Park*,⊥,†,‡ Department of Chemistry, Center for Integrated Molecular Systems, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Korea We report quantitative results on interactions between a tumor suppressor protein, p53, also known as a prognostic cancer marker, and its antibody. The p53 antibody molecules immobilized on an (R)-lipo-diaza18-crown-6 self-assembled monolayer (SAM)-modified gold disk electrode were shown to effectively capture the p53 protein by Western blot, quartz crystal microbalance, and electrochemical impedance experiments. The p53 protein thus captured modulated the ability of the electrode for charge transfer to and from a redox probe in the solution in a p53 concentration range of ∼0.1-30 µg/mL. The same interaction was also observed in the human embryonic kidney cell lysate, demonstrating that the SAM-modified electrode can serve as a selective platform for electrochemically monitoring the cellular p53 concentration. The p53 protein, which has many functions and binding partners in a cell,1 has been intensively studied because of its importance as a cell guardian in cellular proliferation and its tumor suppressing activity. It has also been known that a large number of other genes are regulated by p53.2 The p53 protein also plays a fundamental role in controlling cell growth by activating apoptosis of damaged cells. When p53 is mutated, uncontrolled cell growth usually results in an induction of tumors. It has been reported that more than 50% of human cancers display mutations in the p53 gene.3 Nearly all the mutations lead the gene to be inactivated in the core domain (residues 94-312).4 Due to these functions, a number of scientists have been attempting to assay the p53 protein for an early diagnosis of cancers.5-9 It has been reported that a high nuclear p53 expression level is a significant * Corresponding author. Phone: +82-54-279-2102. Fax: +82-54-279-3399. E-mail: [email protected]. † Department of Chemistry. ‡ Center for Integrated Molecular Systems. § Department of Life Sciences. ⊥ Present Address: School of Energy Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-805, Korea. E-mail: [email protected]. (1) Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307. (2) Ko, L. J.; Prives, C. Genes Dev. 1996, 10, 1054. (3) Levine, A. J. Cell 1997, 88, 323. (4) Olivier, M.; Eeles, R.; Hollstein, M.; Khan, M. A.; Harris, C. C.; Hainaut, P. Hum. Mutat. 2002, 19, 607. (5) Zusman, I.; Sandler, B.; Gurevich, P.; Zusman, R.; Smirnoff, P.; Tendler, Y.; Bass, D.; Shani, A.; Idelevich, E.; Pfefferman, R.; Davidovich, B.; Huszar, M.; Glick, J. Hum. Antibodies Hybridomas 1996, 7, 123.

4770

Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

predictor of the survival of the ovarian cancer cell.9 The elevation of the p53 protein level in blood was also reported in cancer patients.5,6 On the basis of these results, the quantitative determination of the p53 cellular concentration related to its expression level should be a useful prognosis of human cancers. Immunosensors utilizing specific antibody-antigen interactions have been used to selectively detect proteins of different functions.10,11 The quantitative analyses are performed by converting the interaction events on the immunosensors to a measurable signal, such as a fluorescence or current signal.12,13 Though many types of chemically modified surfaces, such as by aldehyde14 and conducting polymers,15 have been made to immobilize biomolecules, these methods had limitations, including the weak adsorption, lower stabilities of proteins, and failures to control the orientation of the immobilized molecules. To overcome these limitations, self-assembled monolayers (SAMs) of thiol derivatives have been suggested as an improvement.16 For instance, bifunctionalized calix[4]crown-5 has been utilized to immobilize protein molecules12 because it captures alkylammonium groups abundant on protein surfaces through host-guest interactions.17 A recent report suggested an improved host, (R)-lipo-diaza-18-crown-6 (hereafter “lipo-diaza crown”).18 The compound not only forms a stable SAM on the gold surface by strongly binding to gold atoms with its four gold-thiol bonds but also provides a better sensitivity for target proteins, enabling their label-free detection to be made (6) Thomas, M. D.; McIntosh, G. G.; Anderson, J. J.; McKenna, D. M.; Parr, A. H.; Johnstone, R.; Lennard, T. W.; Horne, C. H. W.; Angus, B. J. Clin. Pathol. 1997, 50, 143. (7) Levesque, M. A.; Katsaros, D.; Yu, H.; Giai, M.; Genta, F.; Roagna, R.; Ponsone, R.; Massobrio, M.; Sismondi, P.; Diamandis, E. P. Int. J. Cancer 1998, 79, 147. (8) Yu, G.-Q.; Zhou, Q.; Ding, I.; Gao, S.-S.; Zheng, Z.-Y.; Zou, J.-X.; Li, Y.-X.; Wang, L.-D. World J. Gastroenterol. 1998, 4, 365. (9) Psyrri, A.; Kountourakis, P.; Yu, Z.; Papadimitriou, C.; Markakis, S.; Camp, R. L.; Economopoulos, T.; Dimopoulos, M. A. Ann. Oncol. 2007, 18, 709. (10) Bergveld, P. Biosens. Bioelectron. 1991, 6, 55. (11) Luppa, P. B.; Sokoll, L. J.; Chan, D. W. Clin. Chim. Acta 2001, 314, 1. (12) Lee, Y.; Lee, E. K.; Cho, Y. W.; Matsui, T.; Kang, I.-C.; Kim, T.-S.; Han, M. H. Proteomics 2003, 3, 2289. (13) Fu, Z.; Hao, C.; Fei, X.; Ju, H. J. Immunol. Methods 2006, 312, 61. (14) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (15) Cao, T.; Wei, F.; Jiao, X.; Chen, J.; Liao, W.; Zhao, X.; Cao, W. X. Langmuir 2003, 19, 8127. (16) Teramura, Y.; Iwata, H. Anal. Biochem. 2007, 365, 201. (17) De Salvo, G.; Gattuso, G.; Notti, A.; Parisi, M. F.; Pappalardo, S. J. Org. Chem. 2002, 67, 684. (18) (a) Park, J.-Y.; Kim, B. C.; Park, S.-M. Anal. Chem. 2007, 79, 1890. (b) Park, J.-Y.; Lee, Y.-S.; Kim, B. H.; Park, S.-M. Anal. Chem. 2008, 80, 4986. 10.1021/ac900301h CCC: $40.75  2009 American Chemical Society Published on Web 05/12/2009

by electrochemical impedance spectroscopic (EIS) experiments. Consequently, the quantitative analysis of C-reactive protein and ferritin has been performed by EIS in human blood serums.18b The blocking effect caused by the antigen induced the charge transfer resistance of the electrode to increase for an increase in antigen concentrations. Various types of SAMs have been developed for analysis of a variety of biomolecules using similar experimental strategies.19 Efforts to analyze p53 or its antibody,5-9,20 as well as its structures,21,22 have been reported by a few groups. Approaches to determine p53 included high performance liquid chromatography5 and enzyme linked immunosorbent assay based on fluorescence detection.6-8,20 These methods are very sensitive but require tedious procedures5 as well as many chemical steps because the target protein must be labeled by a fluorophore20 or luminescent metal ions must be added to the solution to produce a luminescent complex with the analyte prior to the detection,7 which at times takes as long as a full day or two. Psyrri et al. commented that the immunohistochemistry-based assessment was limited by the nonquantitative nature and introduced what is called an immunofluorescence-based method of automated quantitative measurement for protein analysis.9 In our present work, a p53 antibody immobilized on a lipodiaza crown SAM-modified gold disk electrode was used for the investigation of how the rate of charge transfer of the electrode loaded with p53 molecules on the top of p53 immobilized electrode to and from the redox probe (i.e., Fe(CN)63-/4-) is affected by the p53 protein concentration in the solution. The rate of charge transfer at a given electrode can be measured in the form of its charge transfer resistance. To conserve natural conditions including p53 protein folding, we used the p53 core domain protein as an analyte, which had been expressed molecular biologically in Escherichia coli and purified via ion exchange. The antibody-antigen interaction was then studied by Western blot, quartz crystal microbalance (QCM), and electrochemical impedance spectroscopic experiments. Then the charge transfer resistance was shown to increase linearly with an increase in the p53 concentration in both the electrolyte and real cellular solutions. EXPERIMENTAL SECTION Purification of the p53 Core Domain. The plasmid carrying a DNA sequence encoding the p53 core domain (residues 95-292), which covers the whole DNA binding domain (110-286),21 was provided by Structure Biology of Cancer Laboratory, POSTECH. The plasmid DNA was transformed into the E. coli BL21 (DE3) strain. Its carrying vector was pET-3d. The cells were grown in 1.0 L of Luria-Bertani (LB) media containing 1.5 mM of ampicilin at 310 K until the OD600 (optical density at 600 nm) of the media reached 0.6. Then isopropyl β-D-thiogalactoside (IPTG) was added until its final concentration reached 0.5 mM to induce the p53 gene expression and (19) (a) Lee, J.-Y.; Park, S.-M. J. Phys. Chem. B 1998, 102, 9940. (b) Choi, S.-J.; Choi, B.-G.; Park, S.-M. Anal. Chem. 2002, 74, 1998. (c) Park, J.-Y.; Kwon, S. H.; Park, J. W.; Park, S.-M. Anal. Chim. Acta 2008, 619, 37. (d) Park, J.-Y.; Chang, B.-Y.; Nam, H.; Park, S.-M. Anal. Chem. 2008, 80, 8035. (20) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (21) Che`ne, P. Oncogene 2001, 20, 2611. (22) Wang, Y.; Rosengarth, A.; Luecke, H. Acta Crystallogr., Sect. D 2007, 63, 276.

then incubated at 291 K for 18 h. The cells were harvested and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.2), 50 mM imidazole, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM β-mercaptoethanol while sonicated with a Branson Sonifier. To salt out the target protein, a 10% crystalline (NH4)2SO4 salt in the lysis buffer (w/v) was added to the supernatant solution over a period of 30 min while the solution was mildly stirred. The solution was then neutralized with 0.30 mol of NaOH and stirred for an additional 2 h. The collected precipitate was resuspended in 5 mL of the lysis buffer without DTT, PMSF, and β-mercaptoethanol, followed by dialysis (dialysis membrane with a 12 000-14 000 molecular weight cut-off, Spectrum Laboratories) against 2 L of the same buffer. After removing insoluble materials by centrifuging, the supernatant portion was loaded onto an SP-sepharose cation exchange column (HiTrap SP Sepharose High Performance ion exchange column, GE Healthcare) and eluted with a NaCl gradient eluent from 0 to 600 mM.22 The collected fraction was then dialyzed twice against 2 L of the 10 mM 4-ethylmorpholine buffer (pH 8.5) solution containing 150 mM NaCl to remove imidazole and Tris base. The protein thus obtained was quantified by the Bradford method20,23 using immunoglobulin G (p53 antibody, Abcam) as a standard protein. A calibration curve was constructed by adding a proper volume of immunoglobulin G to a Bradford Reagent (BioRad) and measuring the absorption at 595.5 nm. The concentration of the protein was calculated from the calibration curve. The protein was then diluted to 100 µg/mL with the 10 mM 4-ethylmorpholine buffer (pH 8.5) for use as a stock solution. Western Blot Experiments. The Western blot analysis was carried out to confirm the activity and specificity of the p53 antibody. First, 0.50 µg of the p53 protein was loaded onto a 15% polyacrylamide gel and separated by electrophoresis. The proteins on the gel were transferred to the nitrocellulose membrane (Millipore, Billerica, MA) of a 0.45 µm pore size at 250 mA for 2 h in a transfer buffer containing 20% methanol (v/v), 0.20 M glycine, and 25 mM Tris (pH 7.5). To block nonspecific binding sites, the membrane was incubated in a 20 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 0.10% Tween-20, and 5.0% dry milk (Bio-Rad Laboratories) for 2 h at room temperature. Thereafter, the membrane was incubated sequentially with the primary mouse monoclonal p53 antibody [PAb 240] (Abcam) and the horseradish peroxidase-conjugated secondary antibody to the mouse immunoglobulin (Abcam), each for 1 h. The luminescence of the secondary antibody induced by enhanced chemiluminescent Western blotting detection reagents (Amersham) was then exposed to an X-ray film (AGFA-Gevaert) for 1 min to visualize the protein band where the p53 antibody was bound. Finally, the human embryonic kidney (HEK) cell lysate containing 30 µg of the total protein (7.0 × 106 cells in 1.0 mL of the PBS buffer, Immunology and Disease-Modeling Laboratory, POSTECH) was loaded onto the gel instead of the purified p53, and the same process was repeated to investigate the nonspecific interaction between the p53 antibody and the proteins in the lysate. (23) Stoscheck, C. M. Methods Enzymol. 1990, 182, 50.


4771

Electrochemical Impedance Spectroscopy and Other Experiments. The lipo-diaza crown was synthesized according to the procedure described previously.18b A gold disk electrode (0.020 cm2) was used as a working electrode with Ag/AgCl (in saturated KCl) reference and platinum counter electrodes. The gold electrode was polished with alumina powder of 14.5, 5.0, 1.0, and 0.3 µm until it displayed well-defined cyclic voltammograms in 1 M H2SO4. After drying the surface by purging with N2, the electrode was immersed in 0.10 mM lipo-diaza crown in chloroform for 15 min and washed with a methanolchloroform (1:10) mixed solvent. The modified electrode was then dried with a N2 flow and dipped into a 30 µg/mL p53 antibody solution diluted in a 4-ethylmorpholine buffer (pH 8.5) for 2 h. When the binding was completed, the electrode was washed with the same buffer and dried with N2 again. Impedance data were then obtained in a 10 mM PBS solution containing a redox probe (2.5 mM Fe(CN)63-/4- each) and electrolyte (0.25 M KCl) at an equilibrium potential (0.245 V). The measurements were made using a Solartron model SI 1255 HF frequency response analyzer connected to an EG&G 273 potentiostat/galvanostat by overlaying a small ac wave (±5 mV peak to peak) of frequencies ranging from 100 kHz to ∼100 mHz at 0.245 V. The measurements were stopped when the impedances began to be affected by mass transfer. The data thus obtained were fitted using an appropriate equivalent circuit by the ZsimpWin program (Princeton Applied Research), and charge-transfer resistance (Rct) values were calculated. The electrode modified with both lipo-diaza crown and p53 antibody was then immersed in p53 protein solutions of various concentrations ranging from 0.1 to 50 µg/mL for 2 h. By obtaining the impedance data and calculating Rct values, the dependence of charge transfer resistances on the antigen concentrations was determined. QCM Experiments. A Biomechatron model EQCN 1000 L (Jeonju, Korea) quartz crystal analyzer and a gold-coated AT-cut quartz crystal quartz crystal microbalance (QCM) electrode (area ) 0.196 cm2) with a fundamental frequency of 9.0 MHz were used to monitor the immobilization of antibody and antigen molecules. The lipo-diaza crown SAM was prepared on gold employing the procedure described above. A 1.5 mL aliquot of the 4-ethylmorpholine buffer solution was added into the dried QCM cell containing the SAM-covered QCA electrode. After the frequency was stabilized, a 15 µL aliquot of the 100 µg/mL p53 antibody solution was injected into the cell, and the decrease in frequency was measured. When the frequency stopped decreasing, the buffer was changed and stabilized again. Then the p53 protein was injected to make its final concentration of 50 µg/mL, and the change in frequency was monitored. RESULTS AND DISCUSSION Purification of the Protein and Western Blot Experiments. We first confirmed that the p53 gene cloned in a pET-3d vector did not undergo mutations and contained all residues in the core region, numbers 95-292, properly by sequence analysis. As shown in Figure 1, the core domain protein (22.7 kDa) was strongly expressed in the BL21 (DE3) strain. Although it could be expressed better at 310 K, we set the temperature at 291 K to 4772


Figure 1. The 22.7 kDa p53 core domain expressed in the BL21(DE3) strain. The band between the 21 and 31 kDa regions becomes stronger after the IPTG induction (lane 2) than before the expression (lane 1).

obtain the protein with a more soluble form. Figure 2 shows the improvement of the purity of the target protein by a series of purification steps, including (a) centrifuging, (b) (NH4)2SO4 precipitation, and (c) cation exchange column chromatography. The p53 protein eluted from the SP-sepharose cation exchange column exhibited high enough purity to be used in further experiments. As seen in the chromatogram (Figure 3), the target protein was eluted only when the eluent became electrically conductive. Fraction numbers 4, 5, and 6 were collected and combined. The final protein concentration was determined to be 3.6 mg/mL by the Bradford method after dialysis. The Western blot analysis showed that the purified solutions did not contain any impurities that may bind with the p53 antibody, and the p53 antibody showed a strong binding affinity to the purified target protein (vide infra). In the ∼23 kDa region on lane 1 in Figure 4, a dark band was detected without any other nonspecific bands. QCM Experiments. Immobilization of the p53 antibody and the antigen on a gold-coated QCM electrode modified with the lipo-diaza crown SAM results in an increase in the adsorbed mass on the electrode surface, which is detected by a decrease in the resonance frequency of the quartz crystal electrode. As shown in Figure 5, immobilization of the p53 antibody first on the SAMcovered electrode and the p53 protein subsequently on the antibody-immobilized electrode caused the frequency to decrease to -300 and -210 Hz, respectively. Using the Sauerbrey equation,24 ∆f ) -Cf · m, where the Cf had been obtained to be 2.26 × 102 cm2 · MHz/g by calibration using the silver deposition reaction, the change in weight and the number of bound molecules per unit area were calculated. The number of antibodies (150 kDa) captured on the surface was calculated to be 5.3 × 1012 molecules/cm2, which is in good agreement with the reported theoretical estimation (4.06 × 1012 molecules/cm2).12,18b The amount of antigens bound onto the Fv section of the immobilized antibody molecules was computed to be 2.5 × 1013 molecules/cm2. Thus, the stoichiometric binding (24) Rickert, J.; Brecht, A.; Go ¨pel, W. Anal. Chem. 1997, 69, 1263.

Figure 2. Improvement of the purity of the p53 protein via purification steps, including (a) removal of insoluble materials by centrifuge, (b) (NH4)2SO4 precipitation, and (c) cation exchange column chromatography.

Figure 3. Cation exchange chromatogram. The blue line indicates the UV absorbance of the protein at 280 nm in milli absorbance units due to tyrosine, phenyl alanine, and tryptophan residues. The yellowish green line shows the elutent gradient for the buffer containing 1 M NaCl. The eluate conductivity (cyan color) is shown to increase upon an increase in the NaCl concentration in the elution buffer. Impurities were removed while the eluant conductivity was low and then target protein fractions numbers 4, 5, and 6 were collected at high eluant conductivities.

ratio of antigens to antibody is 4.6:1, which is reasonable considering the smaller molecular weight of the antigen (22.7 kDa)relativetothatoftheantibody(150kDa).Theantigen-antibody binding ratio for the C-reactive protein, which has a molecular weight similar to the p53 core protein (23 kDa), was 5.0:1.12,18b The QCM results clearly verify quantitative deposition of the antibody onto the lipo-diaza crown layer and the quantitative interaction between the immobilized antibody and the antigen. Although the reduction in frequency of the quartz QCM electrode is also dependent on the viscosity of the solution and other parameters, these have been taken care of during the acquisition of the calibration factor (Cf) in the Sauerbrey equation described above. Furthermore, the good agreements of the number of antibody protein molecules obtained with that theoretically

predicted indicate that the measurements do, indeed, represent the correct increase in weight. EIS Measurements. We have reported in a few previous studies18,19 that an increased number of molecules adsorbed on the electrode surface modulates the rate of electron transfer for a redox probe present in the solution, depending on their bulkiness and charges. Specifically, the antigens in solution are captured by the antibodies immobilized on the electrode surface,19d which in turn blocks the charge transfer between the electrode and the redox probe, Fe(CN)63-/4- pair, resulting in an increased charge transfer resistance, Rct. The equivalent circuit was chosen on the basis of a previous description that there would be two routes for electron transfer in a similar Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

4773

Figure 4. Western blot analysis result: a clear band observed in the ∼23 kDa region shows a strong interaction between the p53 antibody and the purified protein when 0.5 µg of the latter was loaded in lane 1. In lane 2, no significant nonspecific interactions were noted when the dialyzed HEK cell lysate containing 30 µg of cellular proteins was loaded.

Figure 5. Decrease in frequency upon injection of (a) 10 µg/mL of the p53 antibody and (b) 50 µg/mL of p53 on the anti-p53 modified surface in the 4-ethylmorpholine buffer (pH 8.5).

situation.19d In contrast to the first path (Rct1 in Figure 6c), which reflects the direct charge transfer to and from the bare gold surface, the second path (Rct2) describes the electron transfer through the immobilized substances, including the SAM, antibodies, and antigens. An identical equivalent circuit was used for analysis of the electrochemical behavior for ferritin and C-reactive proteins interacting with their antibodies at the diaza-crown SAM-modified electrode.18b The total impedance is defined by not only these charge transfer resistances, Rct’s, but also the Warburg impedance (W), solution resistance (Rs), 4774


and the constant phase element (Q).25 The constant phase element is introduced by the SAM-modified surface, which disperses the capacitance of the electrode/electrolyte interface.25a A total resistance for charge transfer is then calculated by a relationship, 1/Rtotal ) 1/Rct1 + 1/Rct2, for the equivalent circuit shown in Figure 6c. The change in impedance was first monitored at an interval of 10 min after the antigen of a known concentration had been injected. We noticed from the experiments that it took about 2 h for the impedance to reach a steady value because of the time required to reach equilibrium for the antibody-antigen reaction. This was also demonstrated by the QCM results showing a frequency plateau about 6000 s after its injection (Figure 5). In addition, the quantity of antigens captured at equilibrium, or the equilibrium total resistance, depended upon the injected antigen concentration. On the basis of these observations, we measured the equilibrium total resistances at various antigen concentrations to establish the dependence between them. As shown in Figure 6a and b, the EIS results showed increasingly larger charge transfer resistances as the antigen concentration was increased. Table 1 shows a few examples of the simulated parameters from the impedance data obtained in a concentration range of ∼0.1-0.9 µg/mL using the ZsimpWin program. As pointed out above, the Rtotal values were obtained from the parallel connection of Rct1 and Rct2; that is, Rct1//Rct2. Even though identical experimental conditions were used to prepare SAMs on a given electrode, the total charge transfer resistances can be significantly different, depending on the batch for SAM preparation due to the variation of morphology and roughness of the electrode surfaces as well as the amounts of the lipo-diaza crown SAM.19c,d,26 For this reason, the total charge transfer resistances obtained in the presence of antigens of various concentrations (Rct,ab+ag) were normalized with respect to that obtained only with its antibodies (Rct,ab) present by dividing the Rct,ab+ag’s by Rct,ab. As shown in Figure 7, the ratio thus obtained is linearly dependent on the p53 concentration. Table 2 also summarizes the Rct values, as well as normalized ratios, for three antigen concentration ranges, ∼0.1-0.9, ∼1.0-9.0, and ∼10-50 µg/ mL. As can be seen from Figure 7 and Table 2, a linear dependence of Rct,ab+ag/Rct,ab on the antigen concentration was observed in the lower concentration region, even though the initial Rct.ab values were not reproducible. Note in Table 2 that the initial Rct,ab values range from as low as 6.36 kΩ to as large as 19.5 kΩ, and yet, the ratios obtained from three different platforms all fall in one line in Figure 7a and b. However, the ratio leveled off when the antigen concentration was >30 µg/mL () 1.3 µM) because all the binding sites of the immobilized antibodies were occupied by antigens. The detection limit estimated from the calibration line is about 0.10 µg/mL () 4 nM). These observations demonstrate that the quantitative analysis of the target protein using its immobilized antibody on the lipo-diaza crown SAM can be made by EIS measurements. (25) (a) Barsoukov, E.; Macdonald, J. R. Impedance Spectroscopy: Theory, Experiment, and Application; Wiley-Interscience: Hoboken, NJ, 2005. (b) Park, S.-M.; Yoo, J.-S. Anal. Chem. 2003, 75, 455A. (c) Park, S.-M.; Yoo, J.-S.; Chang, B.-Y.; Ahn, E.-S. Pure Appl. Chem. 2006, 78, 1069. (d) Chang, B.-Y.; Park, S.-M. Anal. Chem. 2006, 78, 1052. (26) The´venot, D. R.; Toth, K.; Durst, R. A.; Wilson, G. S. Biosens. Bioelectron. 2001, 16, 121.

Figure 6. EIS results obtained for immobilized antibodies and antigens in a solution containing a redox probe (2.5 mM Fe(CN)63-/4- each) and 0.25 M KCl. The p53 concentration range was (a) ∼0.1-0.9 µg/mL and (b) ∼10-50 µg/mL, respectively. (c) Equivalent circuit used for the interpretation of the impedance data. Table 1. Simulated Parameters from the Impedance Data Obtained in ∼0.1-0.9 µg/mL Range

Rs Q n Rct1 Rct2 W Rtotal

anti-p53

p53 (0.1 µg/mL)

p53 (0.3 µg/mL)

p53 (0.5 µg/mL)

p53 (0.7 µg/mL)

p53 (0.9 µg/mL)

68.1 2.02 × 10-6 0.855 1.96 × 104 5.30 × 106 2.55 × 10-4 1.95 × 104

70.1 1.97 × 10-6 0.850 3.29 × 104 4.93 × 107 2.56 × 10-4 3.29 × 104

70.3 1.62 × 10-6 0.863 3.89 × 104 1.36 × 106 2.15 × 10-4 3.78 × 104

70.0 1.56 × 10-6 0.861 4.28 × 104 9.63 × 105 2.05 × 10-4 4.10 × 104

66.6 1.56 × 10-6 0.863 4.28 × 104 1.75 × 106 2.15 × 10-4 4.18 × 104

72.3 1.55 × 10-6 0.857 5.45 × 104 1.89 × 105 2.05 × 10-4 4.23 × 104

p53 Analysis under Physiological Conditions. The p53 core protein dissolved in the HEK cell lysate was quantitatively analyzed to see whether the technique would be applicable to real biological specimens. According to a previous study,27 the nucleic concentration of p53 in normal human cells was on the order of 20 nM, which corresponds to ∼0.45 µg/mL, which may be elevated

significantly when the cells need to be repaired.5 The lysate used in this study contained a small concentration of p53 because the cell extract from only 7.0 × 106 cellular matrixes represents a small fraction of 1 mL of the PBS buffer, which was added to the cell extract. In addition, we found that the immobilized antibodies seemed detached from the lipo-diaza crown SAM Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

4775

Figure 7. The normalized charge transfer resistance vs the p53 core protein concentration in a range of (a) ∼0-50 and (b) ∼0-5 µg/mL. The data in part b are the ones obtained from the HEK cell lysate solutions containing ∼0-10 µg/mL p53. The linear regression was obtained for the data up to 5 µg/mL, with the data obtained above this level discarded. Table 2. The List of Simulated Values of Charge Transfer Resistances and Resistance Ratios for Various Concentrations of p53 Core Protein concentration (µg/mL) Rct,ab, kΩ Rct,ab+ag, kΩ ratio () Rct,ab/Rct,ab+ag) concn, µg/mL Rct,ab, kΩ Rct,ab+ag, kΩ ratio () Rct,ab/Rct,ab+ag) concn, µg/mL Rct,ab, kΩ Rct,ab+ag, kΩ ratio () Rct,ab/Rct,ab+ag)

0.1

0.3

32.9 1.69 1.0

37.8 1.94 3.0

17.3 2.23 10

22.3 3.87 20

36.9 5.8

64.4 10.1

0.5 19.5 41.0 2.11 5.0 7.75 28.3 3.66 30 6.36 89.2 14.0

0.7

0.9

41.8 2.14 7.0

42.3 2.17 9.0

32.7 4.22 40

39.0 5.03 50

97.1 15.3

98.0 15.4

in the cell lysate, resulting in a decrease in the total resistance values for charge transfer, regardless of the antigen concentration due perhaps to the competition between many forms of ammonium ions present in the lysate and the antibodies immobilized for the lipo-diaza crowns. To solve this problem, the cell lysate was first dialyzed in advance against the 4-ethylmorpholine buffer (pH 8.5) without other salts. By eliminating the salts and other interfering substances, which may competitively bind to the lipo-diaza crown SAM, and by making the buffer conditions compatible with our experiments described thus far, we were able to make the measurements of the amounts of antigens in the HEK cell lysate, as was the 4776


case in earlier experiments. Lane 2 in Figure 4 shows that the p53 antibody did not have a significant nonspecific interaction with the proteins present in the dialyzed HEK cell lysate, which was prepared by harvesting 7.0 × 106 lysed cells with the buffer solution to make up its final volume of 1.0 mL. The total concentration of proteins in the lysate solution thus prepared was 30 µg/mL; the protein concentration in the lysate solution was assayed by the Bradford method.20,23 This was also supported by the result that the blank HEK cell lysate solution displayed a resistance ratio of 1.04 without p53 protein added. For the HEK cell lysate solutions containing 13.2 nM to 0.44 µM p53, the resistance ratio varied from 1.62 to 4.91, respectively, which fell in the linear range below 6 µg/mL (Figure 7b). However, the resistance ratio did not increase any more and leveled off beyond the p53 concentration greater than 10 µg/mL (0.44 µM). We believe that aggregations or interactions of many other forms of proteins present in the lysate may also compete with p53 for binding with the p53 antibody in the cell lysate; such a high concentration is not usually found in organisms.27 This set of experiments indicates that p53 in the HEK lysate solutions can be directly analyzed with an appropriate pretreatment without having to separate it prior to its analysis. We conducted a few more experiments to see if other antibodies would also show as strong an affinity as the p53 antibody. We used MSH2 protein and its antibody for this purpose,28,29 and both the Western blot test and impedance measurements did not provide measurable signals, even at relatively high concentrations of MSH2, not to mention p53. We concluded from a series of experiments that the p53 antibody showed a higher specificity for p53 than any other antibodies for not only their corresponding proteins but for p53. Another set of experiments we ran was to use a larger amount of cells obtained from serum such that the cell volume was 1/2 of the final volume with the buffer solution added to see if endogenously present p53 in the cells could be assayed. Ammonium and other small ions were removed by dialysis prior to adding the PBS solution as described above. The result gave 26 (±1.5) nM, corresponding to 0.59 µg/mL from the curve shown in Figure 7b when the p53 level was determined by the standard addition method, in which standard p53 was added such that the added final concentration would be about 1.0 µg/mL. This level is in good agreement with those reported earlier by other investigators,5 which was found to increase to ∼1.7 µg/mL for serums from benign tumor patients and to ∼3.6 µg/mL for malignant cancer patients. CONCLUSIONS In this study, we have demonstrated that the lipo-diaza crown effectively captures the p53 antibody molecules through host-guest interactions, which allows an immunosensor for the selective analysis of the p53 core protein to be constructed. The purified p53 protein with a natural folding structure conserved was used as an analyte to improve the affinity to its antibody and to reproduce biological conditions. To our knowledge, this is the first attempt to immobilize the p53 antibody molecules on any SAMs for label-free detection of the natural p53 protein. We also systematically controlled the experimental conditions such that the well-defined p53 core protein would be obtained for a quantitative study on its interactions with its antibody. The technique developed here is not only sensitive to the change in

antigen concentration but also simple to use. The Western blot and QCM experimental results showed that the interactions between the p53 antibody and p53 are strong enough to make a stable immunosensor. In addition, the method can be easily applied to the analysis of physiological samples without nonspecific interferences when an antibody is chosen appropriately. Although the responses were saturated in a high p53 concentration range, it does not present a serious problem, as a high concentration is not encountered in organic cells. In conclusion, the stability, the sensitivity, and the specificity demonstrate that this is an excellent method to monitor the change in cellular p53 concentration, which can lead to the prognosis of cancers and other cellular malfunctions. ACKNOWLEDGMENT This work was supported by a grant from KOSEF through the Center for Integrated Molecular Systems at Postech (No. R11-

2000-070-06001-0) and by the WCU Program of the Ulsan National Institute of Science and Technology. In addition, graduate stipends were provided by the Korea Research Foundation through its BK21 program. We also thank Ki-Jun Yoon (Immunology and Disease-modeling Laboratory, POSTECH) for preparing and providing the HEK cell lysate. Received for review February 9, 2009. Accepted April 25, 2009. AC900301H (27) Sakaguchi, K.; Sakamoto, H.; Lewis, M. S.; Anderson, C. W.; Erickson, J. W.; Appella, E.; Xie, D. Biochemistry 1997, 36, 10117. (28) Plevova´, P.; Sedla´kova´, E.; Zapletalova´, J.; Krˇepelova´, A.; Sky´palova´, P.; Kola´rˇ, Z. Virchows Arch. 2005, 446, 112. (29) Li, M.; Liu, L.; Wang, Z.; Wang, L.; Liu, Z.; Xu, G.; Lu, S. Oncol. Rep. 2008, 19, 401.


4777

Label-Free Electrochemical Detection of the p53 Core Domain Protein

Recommend Documents