Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Femtomolar level detection of BRCA1 gene using a gold nanoparticle labeled sandwich type DNA sensor P. Abdul Rasheed, N. Sandhyarani ∗ Nanoscience Research Laboratory, School of Nano Science and Technology, National Institute of Technology Calicut, Calicut, Kerala, India
a r t i c l e
i n f o
Article history: Received 15 October 2013 Received in revised form 3 February 2014 Accepted 6 February 2014 Available online 15 February 2014 Keywords: DNA sensor Femtomolar detection BRCA 1 gene STM I–V characteristics Gold nanoparticles.
a b s t r a c t We demonstrate the amplified detection of BRCAI gene based on the gold nanoparticle labeled DNA sensor. The sensor was based on a “sandwich” detection strategy, which involved an immobilized capture probe DNA (DNA-c), Target DNA (DNA-t) and gold nanoparticle conjugated reporter probe DNA (DNAr.AuNP). The sensor surface was characterized by scanning electron microscopy (SEM) and scanning tunneling microscopy (STM). Detection capability of the sensor was studied with I–V measurements using either scanning tunneling microscopy (STM) or Keithley 2400 Source Meter SMU Instrument. The DNA sensor could detect up to 1 fM DNA target (5.896 fg of BRCA 1 gene/ml) and exhibited excellent selectivity against noncomplementary sequences and three base mismatch complementary targets. Good reproducibility, high sensitivity, good stability and reusability of the developed sensor surface showed its application in early cancer diagnosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rapid and sensitive detection of very low concentration of the specific DNA sequences using DNA microarrays or DNA sensors has become an important topic of investigation. In general the sensing surface of a DNA sensor consists of an immobilized single stranded oligonucleotide which binds to its complementary target DNA sequence via hybridization. The hybridization process is then converted into a readable signal by the appropriate transducers such as electrochemical, optical or mass-sensitive elements which can generate readable current, light signals or frequency respectively [1–4]. Biomolecules can easily be conjugated with gold nanoparticles (AuNPs) without losing their biochemical activity. AuNPs are used for signal amplification in several biosensors [5–8] taking advantage of their unique optical and electrical properties. Changes in I–V characteristics are reported as one of the sensitive detection strategies for DNA hybridization event [9]. I–V characteristics can be measured using STM and the gold nanoparticles on the sensor surface give characteristic shape to the I–V plot [10–12]. Electrochemical quartz crystal microbalance (EQCM) is a highly sensitive device to measure change in mass on the surface as the resonant frequency of the crystal changes
∗ Corresponding author. Tel.: +91 495 2286537; fax: +91 495 2287250. E-mail addresses: [email protected], [email protected] (N. Sandhyarani). http://dx.doi.org/10.1016/j.colsurfb.2014.02.009 0927-7765/© 2014 Elsevier B.V. All rights reserved.
with increase in mass on their surface. Hence the EQCM sensors offer sensitive detection of hybridization events even without any optical or redox indicators. This advantage of EQCM was explored for the in-situ monitoring of hybridization as well as fast and less expensive detection [13–16]. BRCA1 (Breast cancer 1) is a human care taker gene that is expressed in the cells of breast and other tissues, where it helps the repair of damaged DNA or destroys the cells, if DNA cannot be repaired. Certain variations of the BRCA1 gene can lead to an increased risk for breast cancer with hereditary breast-ovarian cancer syndrome. Researchers have identified hundreds of mutations in the BRCA1 gene, which are associated with an increased risk of cancer. Mutations in BRCA1 gene are responsible for ∼40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers [17,18]. Hence the sensitive detection of BRCA1 gene mutant in disease-related gene fragments is critical for genetic research and clinical diagnosis of breast cancer [19,20]. In this study, we fabricated a sensitive and simple DNA biosensor for the low concentration detection of BRCA1 DNA (referred as target DNA, DNA-t) sequences. The DNA sensor developed was based on a “sandwich” detection strategy, which involved the immobilization of DNA-c on gold electrode surface. One half of the target DNA is allowed to hybridize to the immobilized DNA-c and the other half to the reporter probe DNA (DNA-r) which is labeled with gold nanoparticles. Such a dual hybridization processes significantly enhanced the signal-to noise ratio, leading to the reliable detection of femtomolar concentrations of DNA targets. In the
8
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
absence of target DNA, the sandwich complexes do not form, leaving the surface-confined capture probe DNA unhybridized. One major advantage of the sandwich type sensor is that no modification is required for the target BRCA1 DNA sequence. Hybridization was monitored as a function of DNA-t concentration using either I–V measurements or measurement of frequency change in QCM. 2. Experimental 2.1. Materials
polished quartz crystal electrode and to monitor the target DNA on the crystal surface. Mass of adsorbed molecules on the surface was calculated using the difference in frequency using Sauerbrey equation [15]. The frequency change with respect to the DNA-t concentration on the EQCM crystal was investigated to evaluate the detection capability of the sandwich type sensing mechanism. The EQCM experiment was performed with a CHI 400A series electrochemical quartz crystal microbalance, (CH Instruments, Texas, USA). 2.6. Functionalization of AuNPs with reporter probe DNA
The oligonucleotides were purchased from Integrated DNA Technologies, USA. The base sequences are as follows: Capture probe (DNA-c): 5 CTT TTG TTC 3 Target probe (DNA-t): 5 GAA CAA AAG GAA GAA AAT C 3 Reporter Probe (DNA-r): 5 GAT TTT CTT C 3 Noncomplementary probe (NC): 5 CCT TGT TGG ACT CCC TTCT 3 Three base mismatch complementary probe (3MM): 5 CAA CAA AAG CAA CAA AAT C 3 Chloroauric acid was purchased from SRL Chemicals, India. O-(3Carboxypropyl)-O -[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol (CPEG), N-(3-Dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride(EDC), 3 sulfo-N-hydroxysuccinimide (NHS) were purchased from Sigma. Other chemicals used were of analytical reagent grade and they were supplied from Sigma and Merck. Ultrapure and deionized water was used in all experiments. Conjugation buffer and hybridization buffer used were 0.1 M NaCl PBS buffer and 0.3 M NaCl PBS buffer respectively [21,22]. 0.1 M NaCl PBS buffer consists of 0.1 M NaCl, 10 mM phosphate buffer (pH 7) and 0.3 M NaCl PBS buffer consist of 0.3 M NaCl, 10 mM phosphate buffer (pH 7). The cleaned Au electrode was used as the sensor surface for I–V and STM I–V measurements. Gold polished quartz crystal electrode was used as the sensor surface for QCM measurements. 2.2. Characterization of sensor surface The sensor surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) with SU-6600 field emission scanning electron microscopy (FESEM, Hitachi, Japan) during each stage of modifications. 2.3. Scanning tunneling microscopy (STM) The STM measurements were performed with the XE-100 STM ((Park Systems, Korea) system under ambient conditions. Both topographic and spectroscopic data were obtained using freshly cut Pt/Ir tips. The topography of the samples was observed with STM, for a set point of 0.1 V and 0.5 nA. The images were captured in each step of hybridization. I–V measurements at various selected points of the sensor surface were also performed in the range of −1.5 to +1.5 V with the same equipment with set points 0.5 nA and 0.1 V at 25 ◦ C, 40–50% humidity. 2.4. I–V measurements I–V characteristics of the sensor were measured by a Keithley 2400 Source Meter SMU Instrument, USA. The electrodes with dimensions 1.2 cm × 0.5 cm was connected to the instrument with a two electrode system. I–V curve was plotted in the potential window of −0.03 V to 0.03 V. 2.5. Electrochemical measurements The electrochemical quartz crystal microbalance (EQCM) was used to calculate the coverage of the DNA molecules on the gold
Gold nanoparticles were synthesized by a monosodium glutamate reduction method as described in the literature [23]. The reduction was done at a pH of 7. The synthesized gold nanoparticles were purified by centrifugation at 12000 rpm for 30 min. Then the precipitate was redispersed in water and treated with 0.05 mM CPEG overnight and purified. Then it was mixed with equimolar mixture (0.5 mM) of EDC and NHS and stirred for 2 h. The unreacted reagents were removed by centrifugation at 12000 rpm for 30 min and repeated washing. Then 100 L of the precipitate was redispersed in 600 L of 0.1 M NaCl PBS buffer. To this mixture, 100 L of 25 M reporter DNA (DNA-r) was added and incubated at 4 ◦ C for 16 h. The unreacted DNA was removed by centrifugation and the precipitate was redispersed in 0.1 M PBS buffer and stored at 4 ◦ C when not in use. These gold nanoparticles functionalized with DNA reporter probe were used in the experiments and denoted as DNA-r.AuNP. 2.7. DNA self-assembly and hybridization at gold electrodes Prior to modification, the working Au electrode was polished with 0.3 m alumina slurry and washed thoroughly with water. The freshly polished electrodes were then pretreated by cleaning with piranha solution followed by sonication in ethanol and water. Afterwards, the Au electrode was electrochemically cleaned by potential cycling for 10 min between 0.2 V and 1.5 V versus Ag/AgCl in 0.1 M perchloric acid (HClO4 ). Self-assembly of the capture DNA probe (DNA-c) on the electrode surface was carried out with the following procedure: the cleaned Au electrode was dipped in 1 M DNA-c probe solution in 0.1 M phosphate buffer (pH 7) for overnight at 4 ◦ C. Then, the electrode was washed with 10 m M PBS solution to remove unbounded DNA strands. This electrode was used throughout the experiment to determine the complementary DNA. The resulted electrode was denoted as Au/DNA-c electrode. The Au/DNA-c electrode was immersed into a solution containing target DNA (DNA-t) in 0.3 M NaCl PBS buffer (pH 7) at 25 ◦ C for 2 h. Subsequently, the electrode was washed to remove nonspecifically adsorbed DNA-t and dried. The complementary portions of the DNA-t were hybridized with DNA-c on the surface, leaving the remaining portion for the hybridization with DNA-r. The hybridization with DNA-r.AuNP was also performed with the same hybridization procedure. Then the resulted Au/DNA-c|DNAt|DNA-r.AuNP electrode was characterized by I–V measurements. To study the effect of gold nanoparticles on the signal enhancement, we conducted the same experiments with DNA-r without any gold nanoparticles. 2.8. Regeneration of the modified electrode For regeneration of the sensor surface the gold electrodes resulted after the complete DNA hybridization (Au/DNA-c|DNAt|DNA-r.AuNP) was immersed in 0.2 M NaCl solution for 10 min followed by washing with 10 mM PBS solution containing 1% SDS [7,24]. Regeneration of the sensor surface was monitored and the reusability of the biosensor surface was tested by repetitive
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
9
Scheme 1. Schematic representations of the various stages of sensor fabrication and DNA hybridization related to BRCA 1.
hybridization with varying target concentrations. For regeneration of gold polished quartz crystal electrode(QCM), the electrode obtained after the complete DNA hybridization (Au/DNA-c|DNAt|DNA-r.AuNP) was immersed in 0.1 M NaOH solution for 10 s followed by washing with 10 mM PBS solution containing 1% SDS [25,26]. 3. Results and discussion The schematic representations of various stages of the sensor fabrication and DNA hybridization related to specific BRCA1 sequences are shown in Scheme 1. Initially, the capture DNA probe (DNA-c) was immobilized on the cleaned gold electrode surface by dipping the electrode in 1 M DNA-c probe solution in 0.1 M phosphate buffer. The resulted electrode was cleaned and then immersed into a solution containing target DNA (DNA-t) in 0.3 M NaCl PBS buffer for 2 h. The complementary portions of the DNA-t were hybridized with DNA-c on the surface, leaving the remaining portion for the hybridization with DNA-r. Followed by this the hybridization with DNA-r.AuNP was achieved. The resulted electrode is denoted as Au/DNA-c|DNA-t|DNA-r.AuNP electrode. 3.1. Characterization of sensor The sensor surface was characterized by scanning electron microscopy and energy dispersive X-ray spectroscopy before and after the hybridization. The SEM images are shown in Fig. 1 and in Fig. S1, Supplementary information. It was seen that the surface became smooth after the immobilization of DNA-c (Fig. S1,
Supplementary information) and further after the hybridization of DNA-t. After hybridization with DNA-r.AuNP, presence of gold nanoparticles on the surface is clearly evident in the SEM image. This confirmed the successful immobilization of DNA-c, hybridization of DNA-t and DNA-r.AuNP on the surface. Size of the gold nanoparticles used for the functionalization of the reporter probe DNA was 15–20 nm, as seen in Fig. 1b. Immobilization of DNA was further confirmed by the presence of nitrogen (1.16 wt%) and phosphorous (1.19 wt%) in the EDS spectrum after the hybridization. To confirm the concentration dependence of DNA-t on the hybridization events we imaged the sensor surface after the hybridization of DNA-r.AuNP in the presence of varying concentrations of DNA-t. It was seen that upon increasing the DNA-t concentration, the number of nanoparticles on the surface increases which directly indicate the increase in concentration of DNA-r and hence the DNA-t on the surface (Fig. S2, Supplementary information). 3.2. Scanning tunneling microscopy The sensor surface was further characterized by scanning tunneling microscopy and monitored the hybridization using I–V curve in STM mode after each hybridization step at 25 ◦ C. The hybridization procedure described in Scheme 1 was used for this experiment. The STM images are shown in Fig. 2. The concentrations of DNA-t and DNA-r.AuNP used in this sensor were 1 pM and 10 nM, respectively. Morphological differences were observed with each steps of modification. The presence of gold nanoparticles was confirmed from the morphology of the surface and the increased current after DNA-r.AuNP hybridization.
Fig. 1. SEM images of the sensor surfaces. a) Au/DNA-c|DNA-t surface and b) Au/DNA-c|DNA-t|DNA-r.AuNP surface. Inset shows magnified images of gold nanoparticles on the surface. 100 pM DNA-t was used for this experiment.
10
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
Fig. 2. The STM images of sensor surfaces in each stage of modification. a) Au surface, b) Au/DNA-c surface, c) Au/DNA-c|DNA-t surface and d) Au/DNA-c|DNA-t|DNA-r.AuNP surface.
STM I–V measurements were performed after the final hybridization in the range of −1.5 to +1.5 V at 25 ◦ C, 40–50% humidity. The set points used for the I–V measurement were 0.5 nA and 0.1 V. The tip–surface distance was adjusted by limiting the approach distance. It was reported that the single stranded DNA (ssDNA) can transport the electrons between the STM tip and the metal surface and the property was higher in double stranded DNA (dsDNA) compared to the ssDNA [27,28]. Any mismatch in the DNA could reflect in the electron transport property. In the present sensor design, after the DNA-r.AuNP hybridization, dsDNA forms on the sensor surface (though with a small defect) which affects the conductivity in a concentration dependent manner. As a result, with the applied voltage, an increase in current with increased concentrations of the DNA-t is expected. STM I–V curves of the sensor surface with varying concentrations of DNA-t (and hence DNA-r.AuNP) are given in Fig. 3. It was seen from the I–V curves that the current increased with increasing concentration of DNA-t. This increase in current is due to the formation of more number of dsDNA and gold nanoparticles on the sensor surface with the increase in concentration of DNA-t. The increase in concentration of DNA-t provided room for more DNA-r.AuNP on the sensor surface and resulted in an increase in the number of nanoparticles on the sensor surface after the hybridization of DNA-r.AuNP. To prove this, SEM images of the sensor surface were taken after the hybridization of DNA-r.AuNP in presence of varying concentrations of DNA-t. The increase in the number of nanoparticles on the sensor surface with increase in concentration of DNA-t was confirmed by the SEM images (Fig. S2, Supplementary information). We observed that the presence of AuNP contributed to an increase in the current compared to the DNA-r alone (Fig. S3, Supplementary information). Detectable change in current was seen up to 1 fM DNA-t and hence 1 fM is the detection limit of the present sensor. The curves were
asymmetric in nature in contrast to the reports because of the discontinuity in the dsDNA structure (there is a chance for a single strand in the DNA-t at the junction of DNA-c and DNA-r). To see the relationship of current with concentration of DNA-t, the current at a potential of 1.4 V is noted for each concentration and the current vs. logarithmic molar concentration of DNA-t is plotted (Fig. 3, inset). It is evident from the figure that the sensor exhibiting a linear relationship between current and logarithmic target concentration in the range of 1 fM to 100 pM BRCA1 DNA.
Fig. 3. The STM I–V curves of sensor with varying concentrations of DNA-t. Inset shows the plot of current vs. logarithmic concentrations of DNA-t. The error bar gives the standard deviation of three repetitive measurements.
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
11
Fig. 4. a) I–V characteristics of the sensor with varying DNA-t concentrations. b) Frequency change (in hybridization with DNA-r.AuNP) with logarithmic concentration of DNA-t. The error bar gives the standard deviation of three repetitive measurements.
3.3. I–V characteristics I–V characteristics of the sensor were measured by the Keithley 2400 Source Meter SMU Instrument, USA. The electrode was connected to the instrument with a two electrode system such that the entire electrode area was available for the measurement. I–V curve was plotted in the potential window of −0.03 V to 0.03 V as this potential window gave constant resistance value in all the measurements. I–V characteristics of the sensor surface with different DNA-t concentrations are given in Fig. 4a. The current increases with increase in concentration of DNA-t which complements the results obtained in STM I–V measurements. The sensor responses were linear in the given range as the whole area of the electrode was accessible for the measurement. However, the STM I–V measurements used only certain points on the sensor surface which led to the nonlinear response. Thus the difference in linearity observed in STM I–V and normal I–V measurements is attributed to the substrate effect. 3.4. EQCM measurements The electrochemical quartz crystal microbalance (EQCM) was used to estimate the mass on the surface and to calculate the coverage of DNA molecules on the sensor surface. Frequency of the quartz crystal was measured during each individual steps of hybridization and the mass of adsorbed molecules on the surface
was calculated from the change in frequency using Sauerbrey equation (Supplementary information). The coverage of DNA molecules on the surface was calculated after immobilizing 1 M DNA-c, after hybridization with 100 pM DNA-t and after second hybridization with 10 nM DNA-r. In this experiment, the DNA-r was not conjugated with AuNP to avoid the complexity of calculation. The frequency change (f) was 8 Hz, 10 Hz and 4 Hz respectively in the three successive modification steps. The coverage was calculated from the f value and the results indicated that 1.09 × 1011 molecules/cm2 of DNA-c, 6.82 × 1010 molecules/cm2 of DNA-t and 5.47 × 1010 molecules/cm2 of DNA-r were present on the sensor surface (see supporting information for the calculation details). The frequency change with respect to the DNA-t concentration of the EQCM crystal was investigated to support the detection capability of the sandwich type sensing mechanism. Here, DNA-r.AuNP was used due to its lower detection limit than the sensor with DNAr alone. The sandwich type detection method as shown in Scheme 1 was adopted in EQCM experiment except the use of gold polished quartz crystal electrode instead of polycrystalline gold electrode. The frequency change was measured in each step of hybridization. The frequency of the crystal before and after the hybridization of DNA-r.AuNP was measured and plotted with respect to the logarithmic concentration of DNA-t in Fig. 4b. Plot showed that the detection limit of the sensor is 100 aM and the frequency change of the sensor exhibited a linear relationship with logarithmic target concentration from 100 aM to 1 nM.
Fig. 5. a) I–V curve showing the regeneration of sensor surface. b) The plot of frequency change with time of the background surface and regenerated surface in EQCM.
12
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
Fig. 6. Selectivity of the sensor surface: comparison of the experiments with complementary target, noncomplementary target and three base mismatch complementary sequences. a) STM I–V curve measurement and b) I–V curve measurement.
3.5. Regeneration of sensor Regeneration of the sensor was achieved by immersing the hybridized electrodes in 0.2 M NaCl solution for 10 min and washing with 10 mM PBS solution. The sensor surface could be regenerated by 96.66 ± .95%. The I–V curve showing the regeneration of sensor surface is given in Fig. 5a. The regeneration of sensor surface was also investigated by EQCM measurements and the results show that the sensor surface was fully regenerated. The plot of change in frequency with time is given in Fig. 5b for the background surface and regenerated surface. 3.6. Selectivity of the sensor surface and the effect of gold nanoparticles Selectivity of the sensor was investigated with noncomplementary (NC) DNA-t and three base mismatch (3MM) complementary DNA-t sequences. 1 pM of DNA-t, NC DNA-t and 3MM DNA-t were used for the experiment. The STM I–V measurements and I–V measurements were used to study selectivity of the sensor surface. The comparison of the experiments with complementary DNA-t, noncomplementary DNA-t and three base mismatch complementary DNA-t sequences is shown in Fig. 6. Hybridization experiments with noncomplementary sequences did not show any change in current from the background current. The experiments with three base mismatch complementary sequences showed a higher current than NC DNA-t and a lesser current than complementary DNA-t. This showed that the present sensor is very selective to complementary sequences and it can be used to detect mismatch sequences. The effect of gold nanoparticles in the signal enhancement was studied by comparing the hybridization with DNA-r alone and DNA-r with AuNP (Fig. S3, Supplementary information). 1 pM DNA-t was used in both the cases and a higher current was observed in the presence of AuNP. This suggested that the sensitivity observed in the developed sensor surface is largely due to the presence of AuNP. I–V measurements also are in agreement with the STM results. 4. Conclusion A simple and robust DNA sensor has been developed for the detection of BRCA1 DNA sequences. SEM and STM measurements indicated that the Au nanostructures and the DNA hybridization are well constructed. We have evaluated the detection limit of the sandwich type DNA sensor using STM I–V, normal I–V and EQCM based methods. This DNA sensor could detect up to 1 fM BRCA1
DNA and exhibited excellent selectivity against noncomplementary sequences and three base mismatch complementary sequences. It was found that the system having AuNPs conjugated to the DNA-r showed enhancement in sensitivity compared with the conventional setup. This DNA sensor showed good reproducibility, stability and reusability. It is expected that the sensor could find applications in cancer biomarker detection in the early stages of cancer, where the concentration of biomarkers is very low.
Acknowledgments The authors acknowledge Department of Science and Technology, Government of India and Council of Scientific and Industrial Research, INDIA for providing financial support. We thank Prof. P. Predeep and his research group at Department of Physics, NIT Calicut for the help they offered for the I–V measurements.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2014. 02.009.
References [1] Q. Luan, Y. Xue, X. Yao, A simple hairpin DNA sensor for label-free detection of sub-attomole DNA target, Sens. Actuators, B 147 (2010) 561–565. [2] X. Su, Y. Wu, W. Knoll, Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization, Biosens. Bioelectron. 21 (2005) 719–726. [3] I˙ . Karamollao˘glu, H.A. Öktem, M. Mutlu, QCM-based DNA biosensor for detection of genetically modified organisms (GMOs), Biochem. Eng. J. 44 (2009) 142–150. [4] X. Liu, Q. Fan, W. Huang, DNA biosensors based on water-soluble conjugated polymers, Biosens. Bioelectron. 26 (2011) 2154–2164. [5] R. Hao, H. Song, G. Zuo, R. Yang, H. Wei, D. Wang, Z. Cui, Z. Zhang, Z. Cheng, X. Zhang, DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection, Biosens. Bioelectron. 26 (2011) 3398–3404. [6] X. Cao, Y. Ye, S. Liu, Gold nanoparticle-based signal amplification for biosensing, Anal. Biochem. 417 (2011) 1–16. [7] A.A. Ensafi, M. Taei, H.R. Rahmani, T. Khayamian, Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode, Electrochim. Acta 56 (2011) 8176–8183. [8] J. Zhang, S. Song, L. Zhang, L. Wang, H. Wu, D. Pan, C. Fan, Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes, J. Am. Chem. Soc. 128 (2006) 8575–8580.
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13 [9] L.A. Jauregui, J.M. Seminario, A DNA sensor for sequencing and mismatches based on electrical transport through Watson–Crick and non Watson–Crick base pairs, IEEE Sens. J. 8 (2008) 803–814. [10] D.M. Ceres, J.K. Barton, In situ scanning tunneling microscopy of DNA-modified gold surfaces: bias and mismatch dependence, J. Am. Chem. Soc. 125 (2003) 14964–14965. [11] J. Qian, S. Liao, M.A. Stroscio, M. Dutta, S. Xu, Electrical transport through single DNA molecules by distinct tip–surface configurations, Proceeding of the 13th International Workshop on Computational Electronics—IWCE 2009, 2009, p. 5091111. [12] H. Cohen, C. Nogues, R. Naaman, D. Porath, Direct measurement of electrical transport through single DNA molecules of complex sequence, PNAS 102 (2005) 11589–11593. [13] J. Wang, P.E. Nielsen, M. Jiang, X. Cai, J.R. Fernandes, D.H. Grant, M. Ozsoz, A. Beglieter, M. Mowat, Mismatch-sensitive hybridization detection by peptide nucleic acids immobilized on a quartz crystal microbalance, Anal. Chem. 69 (1997) 5200–5202. [14] K.A. Marx, Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution–surface interface, Biomacromolecules 4 (2003) 1099–1120. [15] X.C. Zhou, L.Q. Huang, S.F.Y. Li, Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis, Biosens. Bioelectron. 16 (2001) 85–95. [16] S. Hong, H. Jeong, S. Hong, QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV, Talanta 82 (2010) 899–903. [17] C. Li, H. Karadeniz, E. Canavar, A. Erdem, Electrochemical sensing of label free DNA hybridization related to breast cancer 1gene at disposable sensor platforms modified with single walled carbon nanotubes, Electrochim. Acta 82 (2012) 137–142. [18] A. Tutt, A. Ashworth, The relationship between the roles of BRCA genes in DNA repair and cancer predisposition, Trends Mol. Med. 8 (2002) 571–576.
13
[19] H. Xu, L. Wang, H. Ye, L. Yu, X. Zhu, Z. Lin, G. Wu, X. Li, X. Liu, G. Chen, An ultrasensitive electrochemical impedance sensor for a special BRCA1 breast cancer gene sequence based on lambda exonuclease assisted target recycling amplification, Chem. Commun. 48 (2012) 6390–6392. [20] J. Oh, J. Lee, Designed hybridization properties of DNA–gold nanoparticle conjugates for the ultraselective detection of a single-base mutation in the breast cancer gene BRCA1, Anal. Chem. 83 (2011) 7364–7370. [21] J.J. Storhoff, R. Elghanian, C.A. Mirkin, R.L. Letsinger, Sequence-dependent stability of DNA-modified gold nanoparticles, Langmuir 18 (2002) 6666–6670. [22] S. Park, T.A. Taton, C.A. Mirkin, Array-based electrical detection of DNA with nanoparticle probes, Science 295 (2002) 1503–1506. [23] P.R. Chandran, M. Naseer, N. Udupa, N. Sandhyarani, Size controlled synthesis of biocompatible gold nanoparticles and their activity in the oxidation of NADH, Nanotechnology 23 (2012) 015602-1–015602-8. [24] L. Chu, W. Knoll, R. Förch, Plasma polymerized epoxide functional surfaces for DNA probe immobilization, Biosens. Bioelectron. 24 (2008) 118–122. [25] I. Mannelli, M. Minunni, S. Tombelli, R. Wang, M.M. Spiriti, M. Mascini, Direct immobilisation of DNA probes for the development of affinity biosensors, Bioelectrochemistry 66 (2005) 129–138. [26] A. Dupont-Filliard, M. Billon, T. Livache, S. Guillerez, Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film, Anal. Chim. Acta 515 (2004) 271–277. ˚ [27] I. Kratochvílová, K. Král, M. Bunˇcek, A. Víˇsková, S. Neˇspurek, A. Kochalska, T. Todorciuc, M. Weiter, B. Schneider, Conductivity of natural and modified DNA measured by scanning tunneling microscopy. The effect of sequence, charge and stacking, Biophys. Chem. 138 (2008) 3–10. [28] R. Ehlich, J.K.H. Hörber, Molecular order affecting electron transport through ssDNA, Ultramicroscopy 109 (2009) 1074–1079.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Femtomolar level detection of BRCA1 gene using a gold nanoparticle labeled sandwich type DNA sensor P. Abdul Rasheed, N. Sandhyarani ∗ Nanoscience Research Laboratory, School of Nano Science and Technology, National Institute of Technology Calicut, Calicut, Kerala, India
a r t i c l e
i n f o
Article history: Received 15 October 2013 Received in revised form 3 February 2014 Accepted 6 February 2014 Available online 15 February 2014 Keywords: DNA sensor Femtomolar detection BRCA 1 gene STM I–V characteristics Gold nanoparticles.
a b s t r a c t We demonstrate the amplified detection of BRCAI gene based on the gold nanoparticle labeled DNA sensor. The sensor was based on a “sandwich” detection strategy, which involved an immobilized capture probe DNA (DNA-c), Target DNA (DNA-t) and gold nanoparticle conjugated reporter probe DNA (DNAr.AuNP). The sensor surface was characterized by scanning electron microscopy (SEM) and scanning tunneling microscopy (STM). Detection capability of the sensor was studied with I–V measurements using either scanning tunneling microscopy (STM) or Keithley 2400 Source Meter SMU Instrument. The DNA sensor could detect up to 1 fM DNA target (5.896 fg of BRCA 1 gene/ml) and exhibited excellent selectivity against noncomplementary sequences and three base mismatch complementary targets. Good reproducibility, high sensitivity, good stability and reusability of the developed sensor surface showed its application in early cancer diagnosis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Rapid and sensitive detection of very low concentration of the specific DNA sequences using DNA microarrays or DNA sensors has become an important topic of investigation. In general the sensing surface of a DNA sensor consists of an immobilized single stranded oligonucleotide which binds to its complementary target DNA sequence via hybridization. The hybridization process is then converted into a readable signal by the appropriate transducers such as electrochemical, optical or mass-sensitive elements which can generate readable current, light signals or frequency respectively [1–4]. Biomolecules can easily be conjugated with gold nanoparticles (AuNPs) without losing their biochemical activity. AuNPs are used for signal amplification in several biosensors [5–8] taking advantage of their unique optical and electrical properties. Changes in I–V characteristics are reported as one of the sensitive detection strategies for DNA hybridization event [9]. I–V characteristics can be measured using STM and the gold nanoparticles on the sensor surface give characteristic shape to the I–V plot [10–12]. Electrochemical quartz crystal microbalance (EQCM) is a highly sensitive device to measure change in mass on the surface as the resonant frequency of the crystal changes
∗ Corresponding author. Tel.: +91 495 2286537; fax: +91 495 2287250. E-mail addresses: [email protected], [email protected] (N. Sandhyarani). http://dx.doi.org/10.1016/j.colsurfb.2014.02.009 0927-7765/© 2014 Elsevier B.V. All rights reserved.
with increase in mass on their surface. Hence the EQCM sensors offer sensitive detection of hybridization events even without any optical or redox indicators. This advantage of EQCM was explored for the in-situ monitoring of hybridization as well as fast and less expensive detection [13–16]. BRCA1 (Breast cancer 1) is a human care taker gene that is expressed in the cells of breast and other tissues, where it helps the repair of damaged DNA or destroys the cells, if DNA cannot be repaired. Certain variations of the BRCA1 gene can lead to an increased risk for breast cancer with hereditary breast-ovarian cancer syndrome. Researchers have identified hundreds of mutations in the BRCA1 gene, which are associated with an increased risk of cancer. Mutations in BRCA1 gene are responsible for ∼40% of inherited breast cancers and more than 80% of inherited breast and ovarian cancers [17,18]. Hence the sensitive detection of BRCA1 gene mutant in disease-related gene fragments is critical for genetic research and clinical diagnosis of breast cancer [19,20]. In this study, we fabricated a sensitive and simple DNA biosensor for the low concentration detection of BRCA1 DNA (referred as target DNA, DNA-t) sequences. The DNA sensor developed was based on a “sandwich” detection strategy, which involved the immobilization of DNA-c on gold electrode surface. One half of the target DNA is allowed to hybridize to the immobilized DNA-c and the other half to the reporter probe DNA (DNA-r) which is labeled with gold nanoparticles. Such a dual hybridization processes significantly enhanced the signal-to noise ratio, leading to the reliable detection of femtomolar concentrations of DNA targets. In the
8
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
absence of target DNA, the sandwich complexes do not form, leaving the surface-confined capture probe DNA unhybridized. One major advantage of the sandwich type sensor is that no modification is required for the target BRCA1 DNA sequence. Hybridization was monitored as a function of DNA-t concentration using either I–V measurements or measurement of frequency change in QCM. 2. Experimental 2.1. Materials
polished quartz crystal electrode and to monitor the target DNA on the crystal surface. Mass of adsorbed molecules on the surface was calculated using the difference in frequency using Sauerbrey equation [15]. The frequency change with respect to the DNA-t concentration on the EQCM crystal was investigated to evaluate the detection capability of the sandwich type sensing mechanism. The EQCM experiment was performed with a CHI 400A series electrochemical quartz crystal microbalance, (CH Instruments, Texas, USA). 2.6. Functionalization of AuNPs with reporter probe DNA
The oligonucleotides were purchased from Integrated DNA Technologies, USA. The base sequences are as follows: Capture probe (DNA-c): 5 CTT TTG TTC 3 Target probe (DNA-t): 5 GAA CAA AAG GAA GAA AAT C 3 Reporter Probe (DNA-r): 5 GAT TTT CTT C 3 Noncomplementary probe (NC): 5 CCT TGT TGG ACT CCC TTCT 3 Three base mismatch complementary probe (3MM): 5 CAA CAA AAG CAA CAA AAT C 3 Chloroauric acid was purchased from SRL Chemicals, India. O-(3Carboxypropyl)-O -[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol (CPEG), N-(3-Dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride(EDC), 3 sulfo-N-hydroxysuccinimide (NHS) were purchased from Sigma. Other chemicals used were of analytical reagent grade and they were supplied from Sigma and Merck. Ultrapure and deionized water was used in all experiments. Conjugation buffer and hybridization buffer used were 0.1 M NaCl PBS buffer and 0.3 M NaCl PBS buffer respectively [21,22]. 0.1 M NaCl PBS buffer consists of 0.1 M NaCl, 10 mM phosphate buffer (pH 7) and 0.3 M NaCl PBS buffer consist of 0.3 M NaCl, 10 mM phosphate buffer (pH 7). The cleaned Au electrode was used as the sensor surface for I–V and STM I–V measurements. Gold polished quartz crystal electrode was used as the sensor surface for QCM measurements. 2.2. Characterization of sensor surface The sensor surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) with SU-6600 field emission scanning electron microscopy (FESEM, Hitachi, Japan) during each stage of modifications. 2.3. Scanning tunneling microscopy (STM) The STM measurements were performed with the XE-100 STM ((Park Systems, Korea) system under ambient conditions. Both topographic and spectroscopic data were obtained using freshly cut Pt/Ir tips. The topography of the samples was observed with STM, for a set point of 0.1 V and 0.5 nA. The images were captured in each step of hybridization. I–V measurements at various selected points of the sensor surface were also performed in the range of −1.5 to +1.5 V with the same equipment with set points 0.5 nA and 0.1 V at 25 ◦ C, 40–50% humidity. 2.4. I–V measurements I–V characteristics of the sensor were measured by a Keithley 2400 Source Meter SMU Instrument, USA. The electrodes with dimensions 1.2 cm × 0.5 cm was connected to the instrument with a two electrode system. I–V curve was plotted in the potential window of −0.03 V to 0.03 V. 2.5. Electrochemical measurements The electrochemical quartz crystal microbalance (EQCM) was used to calculate the coverage of the DNA molecules on the gold
Gold nanoparticles were synthesized by a monosodium glutamate reduction method as described in the literature [23]. The reduction was done at a pH of 7. The synthesized gold nanoparticles were purified by centrifugation at 12000 rpm for 30 min. Then the precipitate was redispersed in water and treated with 0.05 mM CPEG overnight and purified. Then it was mixed with equimolar mixture (0.5 mM) of EDC and NHS and stirred for 2 h. The unreacted reagents were removed by centrifugation at 12000 rpm for 30 min and repeated washing. Then 100 L of the precipitate was redispersed in 600 L of 0.1 M NaCl PBS buffer. To this mixture, 100 L of 25 M reporter DNA (DNA-r) was added and incubated at 4 ◦ C for 16 h. The unreacted DNA was removed by centrifugation and the precipitate was redispersed in 0.1 M PBS buffer and stored at 4 ◦ C when not in use. These gold nanoparticles functionalized with DNA reporter probe were used in the experiments and denoted as DNA-r.AuNP. 2.7. DNA self-assembly and hybridization at gold electrodes Prior to modification, the working Au electrode was polished with 0.3 m alumina slurry and washed thoroughly with water. The freshly polished electrodes were then pretreated by cleaning with piranha solution followed by sonication in ethanol and water. Afterwards, the Au electrode was electrochemically cleaned by potential cycling for 10 min between 0.2 V and 1.5 V versus Ag/AgCl in 0.1 M perchloric acid (HClO4 ). Self-assembly of the capture DNA probe (DNA-c) on the electrode surface was carried out with the following procedure: the cleaned Au electrode was dipped in 1 M DNA-c probe solution in 0.1 M phosphate buffer (pH 7) for overnight at 4 ◦ C. Then, the electrode was washed with 10 m M PBS solution to remove unbounded DNA strands. This electrode was used throughout the experiment to determine the complementary DNA. The resulted electrode was denoted as Au/DNA-c electrode. The Au/DNA-c electrode was immersed into a solution containing target DNA (DNA-t) in 0.3 M NaCl PBS buffer (pH 7) at 25 ◦ C for 2 h. Subsequently, the electrode was washed to remove nonspecifically adsorbed DNA-t and dried. The complementary portions of the DNA-t were hybridized with DNA-c on the surface, leaving the remaining portion for the hybridization with DNA-r. The hybridization with DNA-r.AuNP was also performed with the same hybridization procedure. Then the resulted Au/DNA-c|DNAt|DNA-r.AuNP electrode was characterized by I–V measurements. To study the effect of gold nanoparticles on the signal enhancement, we conducted the same experiments with DNA-r without any gold nanoparticles. 2.8. Regeneration of the modified electrode For regeneration of the sensor surface the gold electrodes resulted after the complete DNA hybridization (Au/DNA-c|DNAt|DNA-r.AuNP) was immersed in 0.2 M NaCl solution for 10 min followed by washing with 10 mM PBS solution containing 1% SDS [7,24]. Regeneration of the sensor surface was monitored and the reusability of the biosensor surface was tested by repetitive
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
9
Scheme 1. Schematic representations of the various stages of sensor fabrication and DNA hybridization related to BRCA 1.
hybridization with varying target concentrations. For regeneration of gold polished quartz crystal electrode(QCM), the electrode obtained after the complete DNA hybridization (Au/DNA-c|DNAt|DNA-r.AuNP) was immersed in 0.1 M NaOH solution for 10 s followed by washing with 10 mM PBS solution containing 1% SDS [25,26]. 3. Results and discussion The schematic representations of various stages of the sensor fabrication and DNA hybridization related to specific BRCA1 sequences are shown in Scheme 1. Initially, the capture DNA probe (DNA-c) was immobilized on the cleaned gold electrode surface by dipping the electrode in 1 M DNA-c probe solution in 0.1 M phosphate buffer. The resulted electrode was cleaned and then immersed into a solution containing target DNA (DNA-t) in 0.3 M NaCl PBS buffer for 2 h. The complementary portions of the DNA-t were hybridized with DNA-c on the surface, leaving the remaining portion for the hybridization with DNA-r. Followed by this the hybridization with DNA-r.AuNP was achieved. The resulted electrode is denoted as Au/DNA-c|DNA-t|DNA-r.AuNP electrode. 3.1. Characterization of sensor The sensor surface was characterized by scanning electron microscopy and energy dispersive X-ray spectroscopy before and after the hybridization. The SEM images are shown in Fig. 1 and in Fig. S1, Supplementary information. It was seen that the surface became smooth after the immobilization of DNA-c (Fig. S1,
Supplementary information) and further after the hybridization of DNA-t. After hybridization with DNA-r.AuNP, presence of gold nanoparticles on the surface is clearly evident in the SEM image. This confirmed the successful immobilization of DNA-c, hybridization of DNA-t and DNA-r.AuNP on the surface. Size of the gold nanoparticles used for the functionalization of the reporter probe DNA was 15–20 nm, as seen in Fig. 1b. Immobilization of DNA was further confirmed by the presence of nitrogen (1.16 wt%) and phosphorous (1.19 wt%) in the EDS spectrum after the hybridization. To confirm the concentration dependence of DNA-t on the hybridization events we imaged the sensor surface after the hybridization of DNA-r.AuNP in the presence of varying concentrations of DNA-t. It was seen that upon increasing the DNA-t concentration, the number of nanoparticles on the surface increases which directly indicate the increase in concentration of DNA-r and hence the DNA-t on the surface (Fig. S2, Supplementary information). 3.2. Scanning tunneling microscopy The sensor surface was further characterized by scanning tunneling microscopy and monitored the hybridization using I–V curve in STM mode after each hybridization step at 25 ◦ C. The hybridization procedure described in Scheme 1 was used for this experiment. The STM images are shown in Fig. 2. The concentrations of DNA-t and DNA-r.AuNP used in this sensor were 1 pM and 10 nM, respectively. Morphological differences were observed with each steps of modification. The presence of gold nanoparticles was confirmed from the morphology of the surface and the increased current after DNA-r.AuNP hybridization.
Fig. 1. SEM images of the sensor surfaces. a) Au/DNA-c|DNA-t surface and b) Au/DNA-c|DNA-t|DNA-r.AuNP surface. Inset shows magnified images of gold nanoparticles on the surface. 100 pM DNA-t was used for this experiment.
10
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
Fig. 2. The STM images of sensor surfaces in each stage of modification. a) Au surface, b) Au/DNA-c surface, c) Au/DNA-c|DNA-t surface and d) Au/DNA-c|DNA-t|DNA-r.AuNP surface.
STM I–V measurements were performed after the final hybridization in the range of −1.5 to +1.5 V at 25 ◦ C, 40–50% humidity. The set points used for the I–V measurement were 0.5 nA and 0.1 V. The tip–surface distance was adjusted by limiting the approach distance. It was reported that the single stranded DNA (ssDNA) can transport the electrons between the STM tip and the metal surface and the property was higher in double stranded DNA (dsDNA) compared to the ssDNA [27,28]. Any mismatch in the DNA could reflect in the electron transport property. In the present sensor design, after the DNA-r.AuNP hybridization, dsDNA forms on the sensor surface (though with a small defect) which affects the conductivity in a concentration dependent manner. As a result, with the applied voltage, an increase in current with increased concentrations of the DNA-t is expected. STM I–V curves of the sensor surface with varying concentrations of DNA-t (and hence DNA-r.AuNP) are given in Fig. 3. It was seen from the I–V curves that the current increased with increasing concentration of DNA-t. This increase in current is due to the formation of more number of dsDNA and gold nanoparticles on the sensor surface with the increase in concentration of DNA-t. The increase in concentration of DNA-t provided room for more DNA-r.AuNP on the sensor surface and resulted in an increase in the number of nanoparticles on the sensor surface after the hybridization of DNA-r.AuNP. To prove this, SEM images of the sensor surface were taken after the hybridization of DNA-r.AuNP in presence of varying concentrations of DNA-t. The increase in the number of nanoparticles on the sensor surface with increase in concentration of DNA-t was confirmed by the SEM images (Fig. S2, Supplementary information). We observed that the presence of AuNP contributed to an increase in the current compared to the DNA-r alone (Fig. S3, Supplementary information). Detectable change in current was seen up to 1 fM DNA-t and hence 1 fM is the detection limit of the present sensor. The curves were
asymmetric in nature in contrast to the reports because of the discontinuity in the dsDNA structure (there is a chance for a single strand in the DNA-t at the junction of DNA-c and DNA-r). To see the relationship of current with concentration of DNA-t, the current at a potential of 1.4 V is noted for each concentration and the current vs. logarithmic molar concentration of DNA-t is plotted (Fig. 3, inset). It is evident from the figure that the sensor exhibiting a linear relationship between current and logarithmic target concentration in the range of 1 fM to 100 pM BRCA1 DNA.
Fig. 3. The STM I–V curves of sensor with varying concentrations of DNA-t. Inset shows the plot of current vs. logarithmic concentrations of DNA-t. The error bar gives the standard deviation of three repetitive measurements.
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
11
Fig. 4. a) I–V characteristics of the sensor with varying DNA-t concentrations. b) Frequency change (in hybridization with DNA-r.AuNP) with logarithmic concentration of DNA-t. The error bar gives the standard deviation of three repetitive measurements.
3.3. I–V characteristics I–V characteristics of the sensor were measured by the Keithley 2400 Source Meter SMU Instrument, USA. The electrode was connected to the instrument with a two electrode system such that the entire electrode area was available for the measurement. I–V curve was plotted in the potential window of −0.03 V to 0.03 V as this potential window gave constant resistance value in all the measurements. I–V characteristics of the sensor surface with different DNA-t concentrations are given in Fig. 4a. The current increases with increase in concentration of DNA-t which complements the results obtained in STM I–V measurements. The sensor responses were linear in the given range as the whole area of the electrode was accessible for the measurement. However, the STM I–V measurements used only certain points on the sensor surface which led to the nonlinear response. Thus the difference in linearity observed in STM I–V and normal I–V measurements is attributed to the substrate effect. 3.4. EQCM measurements The electrochemical quartz crystal microbalance (EQCM) was used to estimate the mass on the surface and to calculate the coverage of DNA molecules on the sensor surface. Frequency of the quartz crystal was measured during each individual steps of hybridization and the mass of adsorbed molecules on the surface
was calculated from the change in frequency using Sauerbrey equation (Supplementary information). The coverage of DNA molecules on the surface was calculated after immobilizing 1 M DNA-c, after hybridization with 100 pM DNA-t and after second hybridization with 10 nM DNA-r. In this experiment, the DNA-r was not conjugated with AuNP to avoid the complexity of calculation. The frequency change (f) was 8 Hz, 10 Hz and 4 Hz respectively in the three successive modification steps. The coverage was calculated from the f value and the results indicated that 1.09 × 1011 molecules/cm2 of DNA-c, 6.82 × 1010 molecules/cm2 of DNA-t and 5.47 × 1010 molecules/cm2 of DNA-r were present on the sensor surface (see supporting information for the calculation details). The frequency change with respect to the DNA-t concentration of the EQCM crystal was investigated to support the detection capability of the sandwich type sensing mechanism. Here, DNA-r.AuNP was used due to its lower detection limit than the sensor with DNAr alone. The sandwich type detection method as shown in Scheme 1 was adopted in EQCM experiment except the use of gold polished quartz crystal electrode instead of polycrystalline gold electrode. The frequency change was measured in each step of hybridization. The frequency of the crystal before and after the hybridization of DNA-r.AuNP was measured and plotted with respect to the logarithmic concentration of DNA-t in Fig. 4b. Plot showed that the detection limit of the sensor is 100 aM and the frequency change of the sensor exhibited a linear relationship with logarithmic target concentration from 100 aM to 1 nM.
Fig. 5. a) I–V curve showing the regeneration of sensor surface. b) The plot of frequency change with time of the background surface and regenerated surface in EQCM.
12
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13
Fig. 6. Selectivity of the sensor surface: comparison of the experiments with complementary target, noncomplementary target and three base mismatch complementary sequences. a) STM I–V curve measurement and b) I–V curve measurement.
3.5. Regeneration of sensor Regeneration of the sensor was achieved by immersing the hybridized electrodes in 0.2 M NaCl solution for 10 min and washing with 10 mM PBS solution. The sensor surface could be regenerated by 96.66 ± .95%. The I–V curve showing the regeneration of sensor surface is given in Fig. 5a. The regeneration of sensor surface was also investigated by EQCM measurements and the results show that the sensor surface was fully regenerated. The plot of change in frequency with time is given in Fig. 5b for the background surface and regenerated surface. 3.6. Selectivity of the sensor surface and the effect of gold nanoparticles Selectivity of the sensor was investigated with noncomplementary (NC) DNA-t and three base mismatch (3MM) complementary DNA-t sequences. 1 pM of DNA-t, NC DNA-t and 3MM DNA-t were used for the experiment. The STM I–V measurements and I–V measurements were used to study selectivity of the sensor surface. The comparison of the experiments with complementary DNA-t, noncomplementary DNA-t and three base mismatch complementary DNA-t sequences is shown in Fig. 6. Hybridization experiments with noncomplementary sequences did not show any change in current from the background current. The experiments with three base mismatch complementary sequences showed a higher current than NC DNA-t and a lesser current than complementary DNA-t. This showed that the present sensor is very selective to complementary sequences and it can be used to detect mismatch sequences. The effect of gold nanoparticles in the signal enhancement was studied by comparing the hybridization with DNA-r alone and DNA-r with AuNP (Fig. S3, Supplementary information). 1 pM DNA-t was used in both the cases and a higher current was observed in the presence of AuNP. This suggested that the sensitivity observed in the developed sensor surface is largely due to the presence of AuNP. I–V measurements also are in agreement with the STM results. 4. Conclusion A simple and robust DNA sensor has been developed for the detection of BRCA1 DNA sequences. SEM and STM measurements indicated that the Au nanostructures and the DNA hybridization are well constructed. We have evaluated the detection limit of the sandwich type DNA sensor using STM I–V, normal I–V and EQCM based methods. This DNA sensor could detect up to 1 fM BRCA1
DNA and exhibited excellent selectivity against noncomplementary sequences and three base mismatch complementary sequences. It was found that the system having AuNPs conjugated to the DNA-r showed enhancement in sensitivity compared with the conventional setup. This DNA sensor showed good reproducibility, stability and reusability. It is expected that the sensor could find applications in cancer biomarker detection in the early stages of cancer, where the concentration of biomarkers is very low.
Acknowledgments The authors acknowledge Department of Science and Technology, Government of India and Council of Scientific and Industrial Research, INDIA for providing financial support. We thank Prof. P. Predeep and his research group at Department of Physics, NIT Calicut for the help they offered for the I–V measurements.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2014. 02.009.
References [1] Q. Luan, Y. Xue, X. Yao, A simple hairpin DNA sensor for label-free detection of sub-attomole DNA target, Sens. Actuators, B 147 (2010) 561–565. [2] X. Su, Y. Wu, W. Knoll, Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization, Biosens. Bioelectron. 21 (2005) 719–726. [3] I˙ . Karamollao˘glu, H.A. Öktem, M. Mutlu, QCM-based DNA biosensor for detection of genetically modified organisms (GMOs), Biochem. Eng. J. 44 (2009) 142–150. [4] X. Liu, Q. Fan, W. Huang, DNA biosensors based on water-soluble conjugated polymers, Biosens. Bioelectron. 26 (2011) 2154–2164. [5] R. Hao, H. Song, G. Zuo, R. Yang, H. Wei, D. Wang, Z. Cui, Z. Zhang, Z. Cheng, X. Zhang, DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection, Biosens. Bioelectron. 26 (2011) 3398–3404. [6] X. Cao, Y. Ye, S. Liu, Gold nanoparticle-based signal amplification for biosensing, Anal. Biochem. 417 (2011) 1–16. [7] A.A. Ensafi, M. Taei, H.R. Rahmani, T. Khayamian, Sensitive DNA impedance biosensor for detection of cancer, chronic lymphocytic leukemia, based on gold nanoparticles/gold modified electrode, Electrochim. Acta 56 (2011) 8176–8183. [8] J. Zhang, S. Song, L. Zhang, L. Wang, H. Wu, D. Pan, C. Fan, Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes, J. Am. Chem. Soc. 128 (2006) 8575–8580.
P.A. Rasheed, N. Sandhyarani / Colloids and Surfaces B: Biointerfaces 117 (2014) 7–13 [9] L.A. Jauregui, J.M. Seminario, A DNA sensor for sequencing and mismatches based on electrical transport through Watson–Crick and non Watson–Crick base pairs, IEEE Sens. J. 8 (2008) 803–814. [10] D.M. Ceres, J.K. Barton, In situ scanning tunneling microscopy of DNA-modified gold surfaces: bias and mismatch dependence, J. Am. Chem. Soc. 125 (2003) 14964–14965. [11] J. Qian, S. Liao, M.A. Stroscio, M. Dutta, S. Xu, Electrical transport through single DNA molecules by distinct tip–surface configurations, Proceeding of the 13th International Workshop on Computational Electronics—IWCE 2009, 2009, p. 5091111. [12] H. Cohen, C. Nogues, R. Naaman, D. Porath, Direct measurement of electrical transport through single DNA molecules of complex sequence, PNAS 102 (2005) 11589–11593. [13] J. Wang, P.E. Nielsen, M. Jiang, X. Cai, J.R. Fernandes, D.H. Grant, M. Ozsoz, A. Beglieter, M. Mowat, Mismatch-sensitive hybridization detection by peptide nucleic acids immobilized on a quartz crystal microbalance, Anal. Chem. 69 (1997) 5200–5202. [14] K.A. Marx, Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution–surface interface, Biomacromolecules 4 (2003) 1099–1120. [15] X.C. Zhou, L.Q. Huang, S.F.Y. Li, Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis, Biosens. Bioelectron. 16 (2001) 85–95. [16] S. Hong, H. Jeong, S. Hong, QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV, Talanta 82 (2010) 899–903. [17] C. Li, H. Karadeniz, E. Canavar, A. Erdem, Electrochemical sensing of label free DNA hybridization related to breast cancer 1gene at disposable sensor platforms modified with single walled carbon nanotubes, Electrochim. Acta 82 (2012) 137–142. [18] A. Tutt, A. Ashworth, The relationship between the roles of BRCA genes in DNA repair and cancer predisposition, Trends Mol. Med. 8 (2002) 571–576.
13
[19] H. Xu, L. Wang, H. Ye, L. Yu, X. Zhu, Z. Lin, G. Wu, X. Li, X. Liu, G. Chen, An ultrasensitive electrochemical impedance sensor for a special BRCA1 breast cancer gene sequence based on lambda exonuclease assisted target recycling amplification, Chem. Commun. 48 (2012) 6390–6392. [20] J. Oh, J. Lee, Designed hybridization properties of DNA–gold nanoparticle conjugates for the ultraselective detection of a single-base mutation in the breast cancer gene BRCA1, Anal. Chem. 83 (2011) 7364–7370. [21] J.J. Storhoff, R. Elghanian, C.A. Mirkin, R.L. Letsinger, Sequence-dependent stability of DNA-modified gold nanoparticles, Langmuir 18 (2002) 6666–6670. [22] S. Park, T.A. Taton, C.A. Mirkin, Array-based electrical detection of DNA with nanoparticle probes, Science 295 (2002) 1503–1506. [23] P.R. Chandran, M. Naseer, N. Udupa, N. Sandhyarani, Size controlled synthesis of biocompatible gold nanoparticles and their activity in the oxidation of NADH, Nanotechnology 23 (2012) 015602-1–015602-8. [24] L. Chu, W. Knoll, R. Förch, Plasma polymerized epoxide functional surfaces for DNA probe immobilization, Biosens. Bioelectron. 24 (2008) 118–122. [25] I. Mannelli, M. Minunni, S. Tombelli, R. Wang, M.M. Spiriti, M. Mascini, Direct immobilisation of DNA probes for the development of affinity biosensors, Bioelectrochemistry 66 (2005) 129–138. [26] A. Dupont-Filliard, M. Billon, T. Livache, S. Guillerez, Biotin/avidin system for the generation of fully renewable DNA sensor based on biotinylated polypyrrole film, Anal. Chim. Acta 515 (2004) 271–277. ˚ [27] I. Kratochvílová, K. Král, M. Bunˇcek, A. Víˇsková, S. Neˇspurek, A. Kochalska, T. Todorciuc, M. Weiter, B. Schneider, Conductivity of natural and modified DNA measured by scanning tunneling microscopy. The effect of sequence, charge and stacking, Biophys. Chem. 138 (2008) 3–10. [28] R. Ehlich, J.K.H. Hörber, Molecular order affecting electron transport through ssDNA, Ultramicroscopy 109 (2009) 1074–1079.
