Wang Xiaoying Wang Xiaoning Zhang Xiangyi Chen FentianZhu Kehui Yang Ligang Tang Meng

(1Key Laboratory of Environmental Medicine and Engineering of Ministry of Education, Southeast University, Nanjing 210009, China)(2Department of Hematology, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China)

The p53 gene is located on chromosome 17 p13. It contains 11 exons spanning 20 kilobases and encodes a (mostly) nuclear phosphoprotein of 53 kD. This gene belongs to a family of highly conserved genes that contains at least two other members, P63 and P73. However, P53 appears to differ from its cousins by its unique role in tumor suppression. About 50% of all the malignancies contain a mutation in p53 and aggressive growth of several types of cancer has been attributed to mutations in this gene. Moreover, p53 is also involved in sustaining cellular homeostasis and in complex regulatory interactions[1-2]. Sequence-specific analysis of the p53 gene can help early diagnosis of cancer development and consequently increase the success of the treatment[3-4]. Therefore, the specific recognition and quantitative detection of the p53 gene and the mutations in the p53 gene are extremely crucial in fundamental research as well as in clinical practice.

So far, a variety of methods for measuring the p53 gene have been reported. Among traditional methods for molecular diagnosis and also for p53, the distinction is made between point mutation scanning and screening technologies. Scanning technologies aim at finding unknown mutations in candidate or known disease genes, such as direct DNA sequencing. Screening techniques with high throughput[5], such as denaturing high-performance liquid chromatography (DHPLC)[6], single-strand conformation polymorphism (SSCP)[7], and denaturing gradient gel electrophoresis (DGGE)[8],aim at finding known mutations. However, some of these approaches are time-consuming and require highly skilled labor, while others are less sensitive or more expensive in equipment use. In recent years, a new trend for the detection of p53 mutations has turned to high-sensitivity and specificity, real-time and rapid detection. Biosensors, in particular DNA-based sensors, are of considerable recent interest due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared with traditional hybridization assays. Different transduction principles have been employed for p53 DNA detection including electrochemical[9-13], piezoelectric[14-15]and optical techniques[16-18].

1 Materials and Methods

1.1 Reagents and apparatus

ECL was recorded with a MPI-E electrogenerated chemiluminescence analyzer (Xi’an Remax Electronic Science-Tech Co., Ltd., China), and a CHI 660A electrochemical analyzer (CHI instruments Inc., USA) was used to measure impedance and cyclic voltammogram (CV) in a 10 mL analytical cell.

1.2 Fabrication of ADH coexisted solid-state ECLbiosensor

Fig.1 Schematic representation of the preparation of ADH-coexisted solid-state ECL biosensor for detection of p53 sequences

The ssDNA electrode was immersed into 10 mmol/L PBS (pH 7.3) containing 0.5 mol/L NaCl and AuNPs-labeled p53 probe (AuNPs-labeled p53 probe was prepared according to Ref.[19]), and a constant potential at+0.5 V (vs. Ag/AgCl) was applied for 300 s. Then the electrode was washed thoroughly with 10 mmol/L PBS (pH 7.3) to remove the unhybridized probe. The AuNPs-dsDNA electrode was obtained.

The AuNPs-dsDNA electrode was incubated with ADH in 10 mmol/L PBS (pH 7.3) at 4℃ for 12 h to attach ADH onto AuNPs. Then the electrode was washed with the same buffer thoroughly. The ADH electrode was obtained and employed as working electrode to detect ECL signal.

1.3 Preparation of real sample

The soluble cell lysates of the GES-1 normal gastric mucosal cells and MGC-803 gastric cancer cells were prepared according to Makmura et al.[20].The procedure is as follows:Cells were washed three times with ice-cold 10 mmol/L PBS containing 137 mmol/L NaCl and 2.7 mmol/L KCl (pH 7.4). After decanting the PBS solution, cells were lysed in 50 mmol/L Tris-HCl containing 150 mmol/L NaCl, 0.02% NaN3, 0.1% sodium dodecyl sulfate (SDS), 100 μg/mL phenylmethanesulfonyl fluoride (PMSF), 1 μg/mL aprotinin, 1% Triton X-100 and 0.5% sodium deoxycholate (pH 8.0) on ice for 20 min. The lysed cells were then removed from the tube walls by a cell slicker and transferred to a centrifuge tube. After sonication for 30 s on ice, contents released from the cell were centrifuged at 4 ℃ at 12 000 r/min for 10 min. The supernatant was collected and mixed with a fresh AuNPs solution for 40 h at 4 ℃. The mixtures were stored at 4 ℃ for later use.

1.4 ECL measurement

The ECL determinations were performed at room temperature in a 10 mL homemade quartz cell. A three-electrode system used in this study included the modified GC electrode (3 mm in diameter) as the working electrode, an Ag/AgCl (sat.) as the reference electrode and a platinum wire as the counter electrode. The cyclic voltammetry mode with continuous potential scanning from 0 to 1.2 V and the scanning rate of 0.1 V/s was applied to achieve the ECL signal in 20 mmol/L PBS containing 300 μmol/L alcohol and 1.0 mmol/L NAD+(pH 7.5). Since NAD+was unstable in a strongly alkaline solution[21], pH 7.5 was used in all experiments. A high voltage of-800 V was supplied to the photomultiplier for luminescence intensity determination. The ECL and CV curves were recorded simultaneously.

2 Results and Discussion

2.1 SEM images of COOH-MWNTs, MWNTs-Ru(bpy) composite and MWNTs-Ru(bpy)-PPy electrode

Fig.2 SEM images. (a) COOH-MWNTs; (b) MWNTs-Ru(bpy) composites; (c) MWNTs-Ru(bpy)-PPy electrode

2.2 Characterization of ADH-coexisted solid-state ECL biosensor

Fig.3 The characterization of ADH-coexisted solid-state ECL biosensor. (a) Nyquist plots for the impedance measurement in 10 mmol/L [Fe(CN)6]3-/4- solution; (b) The corresponding cyclic voltammogram curves in 1 mmol/L [Fe(CN)6]3-/4- solution

2.3 Optimization of experimental conditions

The detecting solution has a great impact on ECL intensity. The signal of the influence of NAD+concentration was investigated. In 20 mmol/L PBS (pH 7.5), with the concentration of NAD+increasing, the ECL intensity increased. When 1.5 mmol/L NAD+were added, the response reached the largest. As the concentration kept increasing, the signal decreased slightly. The increased response may be explained by higher conversion efficiency with higher concentration of NAD+in the enzyme-catalyzed reaction. Considering the high cost of cofactor NAD+, 1.0 mmol/L NAD+was used in all other experiments. Furthermore, 20 mmol/L PBS (pH 7.5) containing 300 μmol/L ethanol can provide stable ECL signal. Therefore, 20 mmol/L PBS containing 300 μmol/L alcohol and 1.0 mmol/L NAD+(pH 7.5) was selected as the detecting solution.

2.4 Specificity, repeatability and stability of ADH-coexisted solid-state ECL biosensor

Fig.4 ECL intensity of ssDNA electrodes hybridization with different sequences

The ECL intensity of the solid-state ECL biosensor is recorded under continuously cyclic potential scanning for 20 cycles in 20 mmol/L PBS containing 300 μmol/L alcohol and 1.0 mmol/L NAD+(pH 7.5) at a scan rate of 0.1 V/s. There was no obvious change in the ECL signal. Even after hundreds of cycles, only a slight decrease in the ECL signal was observed, indicating the good stability of the ADH-coexisted solid-state ECL biosensor.

2.5 Calibration curve of wtp53 detection

The sensitivity of the solid-state ECL biosensor is investigated. Fig.5(a) shows theIECL′(the difference of ECL intensity between the ADH electrode and AuNPs-dsDNA electrode) of the ADH electrodes under the conditions that different concentrations of AuNPs-labeled wtp53 interact with the ssDNA.IECL′is grown when the AuNPs-labeled wtp53 concentration is increased.IECL′is found to be linear with the logarithm of the wtp53 concentration in the range from 0.3 to 300 pmol/L (containing 0.3, 3, 30 and 300 pmol/L) in Fig.5(b). The equation for the resulting calibration plot isy=84.6lgx+90.1 wherexis the concentration of wtp53 andyisIECL′; the correlation coefficient is 0.9987, and a detection limit of 0.1 pmol/L is estimated by using 3σ, whereσis the relative standard deviation of a blank solution, andn=11. Meanwhile, the signal of the mtp53 (C/T mismatched) turns out to be about 57.1% that of the wtp53 when they are in the same concentration (see Fig.5(b)).Therefore, the enzyme-based ECL sensing platform can recognize sequence-specific p53 sequences (wtp53 and mtp53) with a discrimination of up to 57.1%. The consistent data is obtained as shown in Fig.5(b) when the experiment is repeated three times.

Fig.5 Calibration curve of p53 detection. (a) ECL intensity-potential curves for the ADH electrodes with various wtp53 concentrations; (b) Calibration curves of p53 detection

2.6 Real sample analyses

Finally, we explored the feasibility of the method for real sample analyses. AuNPs were attached to the cysteine residues on the p53 molecules in the soluble cell lysates of the GES-1 normal gastric mucosal cells and the MGC-803 gastric cancer cells. Then, the ssDNA recognized the AuNPs-labeled p53 gene, and the AuNPs layer adsorbed the ADH molecules for producing the ECL signal. A well-defined ECL peak was observed when the ssDNA electrodes were used to capture wtp53 from the GES-1 normal gastric mucosal cell lysates. Meanwhile, the signal of the wtp53 in the MGC-803 gastric cancer cell lysates turns out to be about 61.8% that of the wtp53 in the GES-1 normal gastric mucosal cell lysates when they are in the same experimental conditions. To further establish the validity of this method for clinical applications, we conducted ELISA tests in parallel with this method for the analyses of the soluble cell lysates of the GES-1 normal gastric mucosal cells and the MGC-803 gastric cancer cells. As shown in Tab.1, the significantly higher total p53 concentration in cancer cell lysates, determined by the ELISA test, is predominantly contributed by the elevation of the mutant p53. When compared with the results obtained with our method, it is clear that the elevation of the mutant p53 concentration is accompanied by a precipitous decline of the wtp53 concentration. Remarkably, the cancer cell assayed by our method displayed substantially (about 59 times, calculated according to the calibration curve of wtp53) lower wtp53 concentrations than that in the normal cell lysates, suggesting that the p53 gene had been severely mutated in these MGC-803 gastric cancer cells. These results were consistent with Ref.[12]. The consistent data was obtained when the experiment was repeated three times. The data in Tab.1 demonstrate that our method is highly complementary to ELISA. The method is capable of determining p53 from real samples without extensive sample pretreatment/separation or specialized instruments and does not require the use of p53 antibodies. It holds promise as a clinical protocol for assaying p53 DNA binding capacity in normal and cancer cells at sensitive levels.

Tab．1 Comparisons of total p53 and wtp53 concentrations between cancer and normal cell lysates nmol/L

3 Conclusion

[1]Hofseth L J, Hussain S P, Harris C C. p53: 25 years after its discovery [J].TrendsPharmacolSci, 2004,25(4): 177-181.

[2]Vousden K H, Lane D P. p53 in health and disease [J].NatRevMolCellBiol, 2007,8(4): 275-283.

[3]Hainaut P, Wiman K G. 30 years and a long way into p53 research [J].LancetOncol, 2009,10(9): 913-919.

[4]Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use [J].ColdSpringHarbPerspectBiol, 2010,2(1): a001008-01-a001008-17.

[5]Jiang T, Minunni M, Mascini M. Towards fast and inexpensive molecular diagnostic: the case of p53 [J].ClinChimActa, 2004,343(1/2): 45-60.

[6]Narayanaswami G, Taylor P D. Site-directed mutagenesis of exon 5 of p53: purification, analysis, and validation of amplicons for DHPLC [J].GenetTest, 2002,6(3): 177-184.

[7]Miyajima K, Tamiya S, Oda Y, et al. Relative quantitation of p53 and MDM2 gene expression in leiomyosarcoma; real-time semiquantitative reverse transcription-polymerase chain reaction [J].CancerLett, 2001,164(2): 177-188.

[8]Van Orsouw N J, Dhanda R K, Rines R D, et al. Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests [J].NucleicAcidsRes, 1998,26(10): 2398-2406.

[9]Wang J X, Zhu X, Tu Q Y, et al. Capture of p53 by electrodes modified with consensus DNA duplexes and amplified voltammetric detection using ferrocene-capped gold nanoparticle/streptavidin conjugates [J].AnalChem, 2008,80(3):769-774.

[10]Zhou H J, Xing D, Zhu D B, et al. Rapid and sensitive detection of point mutation by DNA ligase-based electrochemiluminescence assay [J].Talanta, 2009,78(4/5): 1253-1258.

[11]Gupta G, Atanassov P. Electrochemical DNA hybridization assay: enzyme-labeled detection of mutation in p53 gene [J].Electroanalysis, 2011,23(7): 1615-1622.

[12]Farjami E, Clima L, Gothelf K, et al. “Off-on” electrochemical hairpin-DNA-based genosensor for cancer diagnostics [J].AnalChem, 2011,83(5):1594-1602.

[13]Raoofa J B, Ojania R, Golabib S M, et al. Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide[J].SensorActuatBChem, 2011,157(1): 195-201.

[14]Han S H, Kim S K, Park K, et al. Detection of mutant p53 using field-effect transistor biosensor [J].AnalChimActa, 2010,665(1):79-83.

[15]Chen C P, Ganguly A, Lu C Y, et al. Ultrasensitive in situ label-free DNA detection using a GaN nanowire-based extended-gate field-effect-transistor sensor[J].AnalChem, 2011,83(6): 1938-1943.

[16]Jiang T S, Minunni M, Wilson P, et al. Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor [J].BiosensBioelectron, 2005,20(10): 1939-1945.

[17]Wang Y C, Zhu X, Wu M H, et al. Simultaneous and label-free determination of wild-type and mutant p53 at a single surface plasmon resonance chip preimmobilized with consensus DNA and monoclonal antibody [J].AnalChem, 2009,81(20): 8441-8446.

[18]Qiu L P, Wu Z S, Shen G L, et al. Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence [J].AnalChem, 2011,83(8): 3050-3057.

[19]Wang X Y, Zhang X Y, He P G, et al. Sensitive detection of p53 tumor suppressor gene using an enzyme-based solid-state electrochemiluminescence sensing platform [J].BiosensBioelectron, 2011,26(8): 3608-3613.

[20]Makmura L, Hamann M, Areopagita A, et al. Development of a sensitive assay to detect reversibly oxidized protein cysteine sulfhydryl groups antioxid [J].AntioxidRedoxSign, 2001,3(6): 1105-1118.

[21]Park J K, Yee H J, Lee K S, et al. Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase[J].AnalChimActa, 1999,390(1/2/3): 83-91.

[22]Cai H, Lee T M H, Hsing I M. Label-free protein recognition using an aptamer-based impedance measurement assay [J].SensorActuatBChem, 2006,114(1): 433-437.

[23]Li Y, Qi H L, Peng Y G, et al. Electrogenerated chemiluminescence aptamer-based method for the determination of thrombin incorporating quenching of tris (2,2′-bipyridine) ruthenium by ferrocene[J].ElectrochemCommun, 2008,10(9): 1322-1325.