Biosensors and Bioelectronics 65 (2015) 333–340

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Attomolar detection of BRCA1 gene based on gold nanoparticle assisted signal amplification P Abdul Rasheed, N Sandhyarani n Nanoscience Research Laboratory, School of Nano Science and Technology, National Institute of Technology Calicut, Calicut, Kerala, India

art ic l e i nf o

a b s t r a c t

Article history: Received 22 July 2014 Received in revised form 16 October 2014 Accepted 17 October 2014 Available online 30 October 2014

In this work, we report a simple strategy for signal amplification using appropriately functionalized gold nanoparticles in an electrochemical genosensor which led to attomolar detection of breast cancer 1 (BRCA1) gene. The sensor was developed by the layer-by-layer assembly of mercaptopropionic acid (MPA), polyethylene glycol (PEG) functionalized gold nanoparticle (AuNPPEG), capture DNA (DNA-c), target BRCA1 DNA (DNA-t) and gold nanoparticle labeled reporter DNA (DNA-r.AuNP) on gold electrode. PEG functionalized gold nanoparticles on the MPA surface provided good electron conducting path nullifying the insulating effect of MPA and also act as a proper immobilization platform for the DNA-c by the large number of carboxyl groups present on the functionalized gold nanoparticles. We demonstrated that the incorporation of MPA functionalized gold nanoparticles (AuNPMPA) as an electrochemical label in this sensor design could significantly enhance the sensitivity in the detection. The DNA hybridization of DNA-r.AuNP with target probe was measured by chronoamperometry, electrochemical impedance spectroscopy (EIS), and scanning tunnelling spectroscopy (STS). Electrochemical quartz crystal microbalance (EQCM) experiments were used to support the detection and also to calculate the number of adsorbed molecules on the surface. Under optimum conditions the present sensor exhibited high sensitivity and a very low detection limit of 50 attomolar DNA target (294.8 attogram BRCA1 gene/ml). It shows excellent selectivity against non complementary sequences and 3 base mismatch complementary targets. It also shows good reproducibility, stability and reusability and the developed sensor surface is suitable for point-of care applications. & Elsevier B.V. All rights reserved.

Keywords: Genosensor BRCA1 gene Functionalized gold nanoparticles Chronoamperometry Scanning electron spectroscopy EQCM

1. Introduction Development of genosensors for the sensitive detection of different genes is of high priority in the diagnosis of genetic diseases, mutation detection and genomic sequencing. (Hianik et al., 2001; Zhu et al., 2011; Cai et al., 2002; Lao et al., 2005). In a genosensor, the hybridization process between DNA and its complementary sequences is converted into a readable signal by appropriate transducers like electrochemical, surface plasmon resonance, optical or mass-sensitive elements (Luan et al., 2010; Su et al., 2005; Karamollaoğlu et al., 2009; Liu et al., 2011). Among the different genosensors, electrochemical genosensors offer great promise for DNA sensing due to their high sensitivity, selectivity, low cost and ease of miniaturization (Wang, 2006; Sadik et al., 2009). The main process in the fabrication of DNA electrochemical biosensors is to prepare well defined DNA recognition interface by effective immobilization of capture probe DNA onto the transducer n

Corresponding author. Fax: 91 495 2287250. E-mail address: [email protected] (N. Sandhyarani).

http://dx.doi.org/10.1016/j.bios.2014.10.054 0956-5663/& Elsevier B.V. All rights reserved.

surface through suitable matrix and immobilization method. Various methods such as covalent binding, biotin-avidin interaction, controlled potential adsorption and entrapment in a polymer matrix have been used to immobilize DNA onto the electrode surface (He et al., 2005; Lucarelli et al., 2008). Self-assembled monolayer (SAM) is a commonly used strategy for linking biomolecules to the surface. (Satjapipat et al., 2001; Chaki and Vijayamohanan, 2002; Peeters and Stakenborg, 2010). Gold nanoparticles (AuNPs) have been widely used as agents for signal amplification in electrochemical sensors. (Cao et al., 2011; Ensafi et al., 2011). DNA–gold nanoparticle conjugates (DNA–AuNPs) have been investigated as one of the most attractive nanomaterials utilized for a number of powerful and versatile diagnostic applications (Oh and Lee, 2011; Niemeyer and Simon, 2005). Breast cancer 1 (BRCA1) gene is a human cancer suppresser gene that helps to repair damaged DNA. Mutations in BRCA1 gene are responsible for the increased risk of breast and ovarian cancers (Tutt and Ashworth, 2002; Li et al., 2012). Hence detection of Breast cancer 1 (BRCA1) gene and its mutants in disease-related gene fragments are critical for genetic research and clinical diagnosis of breast cancer (Xu et al., 2012; Oh and Lee, 2011).

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In this work, mercaptopropionic acid (MPA) and functionalized gold nanoparticles (AuNPPEG) were immobilized on the gold electrode surface (Au electrode) before the immobilization of DNA-c. The sensor relies on the hybridization of the probes with their complementary target which are immobilized on the electrode. The DNA probes are hybridized by “sandwich” hybridization scheme, which involves capture probe DNA on the AuNPPEG hybridize to one half of the target DNA and reporter probe DNA labeled with gold nanoparticles hybridize to the other half of target DNA. We compared the effect of functionalization of AuNP using AuNP functionalized with cyclic bisureas (AuNPCBU), AuNP functionalized with citrate (AuNPcitrate) and AuNPPEG. It was observed that maximum number of DNA-c was immobilized on AuNPPEG and hence leads to higher sensitivity. Incorporation of another gold nanoparticle as electrochemical label significantly enhanced the sensitivity in the electrochemical detection due to the efficient electron conduction of gold nanoparticles. Since the electron transfer property is a function of chain length (Mathew and Sandhyarani, 2014) we have used the gold nanoparticles functionalized with mercaptopropionic acid (AuNPMPA) as label to achieve maximum electron conduction. The highest sensitivity obtained for the sensor is greatly due to the presence of two conducting centers (gold nanoparticles) in the sensor design.

2. Experimental methods 2.1. Materials The oligonucleotides were purchased from Integrated DNA Technologies, USA. The base sequences are as follows Target probe (DNA-t): 5′ GAA CAA AAG GAA GAA AAT C 3′ Capture probe (DNA-c): 5′ CTT TTG TTC 3′ Reporter Probe (DNA-r): 5′ GAT TTT CTT C 3′ Three base mismatch complementary probe (3MM): 5′ CAA CAA AAG CAA CAA AAT C 3′ Non complementary probe (NC): 5′CCT TGT TGG ACT CCC TTC T 3′ HAuCl4  3H2O was purchased from SRL chemicals India. O-(3-Carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol (Carboxy PEG), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(EDC), 3 sulfo-N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich, India. Mercaptopropionic acid (MPA) was purchased from Acros organics, India. Other chemicals used were of analytical reagent grade and they were supplied from Himedia and Merck, India. Ultrapure and deionized water was used in all experiments. Conjugation buffer and hybridization buffer used were 0.1 M PBS and 0.3 M PBS respectively (Storhoff et al., 2002; Park et al., 2002). 0.1 M PBS is 10 mM PBS (pH7) with 0.1 M NaCl and 0.3 M PBS 10 mM PBS (pH7) with 0.3 M NaCl. 2.2. Measurement methods Cyclic voltammetry (CV), and chronoamperometry measurements were performed with a CHI 400 A electrochemical analyzer (CH Instruments, Texas, USA). A three-electrode system was used with Pt wire as auxiliary electrode, calomel electrode as reference electrode, and modified gold electrode as the working electrode. Cyclic voltammetric measurements were carried out at room temperature in 10 mM PBS buffer with a scan rate of 0.1 V/s. Chronoamperometric measurements were done at an applied potential of  0.35 V. Electrochemical impedance spectroscopy (EIS) measurements were carried out with a Gill AC computer controlled electrochemical workstation (ACM, U.K model no: 1475). The measurements were taken by conventional threeelectrode configuration with platinum sheet (1 cm2 surface area) as auxiliary electrode, modified gold electrode as the working

electrode and saturated calomel electrode (SCE) as the reference electrode. The working electrode was first immersed in the test solution and after establishing a steady state open circuit potential, the electrochemical measurements were carried out with amplitude of 10 mV ac sine wave with a frequency range of 0.01 Hz–10,000 Hz. The electrochemical quartz crystal microbalance (EQCM) experiment was performed using an EQCM attachment with CHI 400 A electrochemical analyzer. EQCM experiments were carried out on a modified gold polished quartz crystal electrode of resonance frequency 7.9 MHz as working electrode, calomel electrode as reference electrode and a platinum wire as counter electrode. Scanning electron microscope (SEM) images were obtained using Hitachi SU6600 variable pressure field emission scanning electron microscope (FESEM, Hitachi, Japan). Atomic force microscopy images were obtained from XE-100 atomic force microscope (Park systems, Korea). Scanning tunnelling microscopy (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 at each step of hybridization. Scanning tunnelling spectroscopy measurements at various selected points of the sensor surface were performed in the range of  1.5 to þ1.5 V with the same equipment with set points 0.5 nA and 0.1 V at 25 °C, 40–50% humidity. 2.3. Synthesis of AuNPPEG Gold nanoparticles were synthesized by glutamate reduction method according to a reported procedure (Chandran et al., 2012). The synthesized gold nanoparticles were purified by centrifugation at 12,000 rpm for 30 min. The precipitate was redispersed in carboxy PEG and kept overnight. The unreacted substances were removed by centrifugation at 12,000 rpm for 30 min several times. The final product obtained after centrifugation was redispersed in conjugation buffer and it is denoted as AuNPPEG. Size of the nanoparticles thus obtained was 20–25 nm. 2.4. Synthesis of AuNPMPA and functionalization of AuNPMPA with DNA-r The gold nanoparticles synthesized by the glutamate reduction procedure were functionalized with MPA by stirring a mixture of 5 ml gold nanoparticle solution in water and 1 ml 100 μM MPA for 3 h. After purification by centrifugation at 12,000 rpm for 30 min, 100 mL of the precipitate was redispersed in 600 mL of 0.1 M PBS buffer and it is referred as AuNPMPA. To this AuNPMPA, 100 mL of 25 mM reporter DNA (DNA-r) was added and incubated at room temperature 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. These gold nanoparticles functionalized with DNA reporter probe were used in the experiments and denoted as DNA-r.AuNPMPA. Here the DNA is bonded to MPA through hydrogen bonding between the carboxyl group of MPA and cytosine or guanine. To evaluate the effect of chain length of the stabilizer in the sensitivity of the sensor, we have conducted experiments with DNA-r.AuNPPEG. The conjugation of DNA-r with AuNPPEG was done by the reported procedure (Rasheed and Sandhyarani, 2014). We observed that AuNPMPA gives higher sensitivity and hence only that is reported here.

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2.5. Sensor fabrication Prior to modification, the working Au electrode was polished with 0.3 mm alumina slurry and washed thoroughly with water. The freshly polished electrode was then cleaned 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 (HOCl4). Mercaptopropionic acid (MPA) monolayer was prepared by dipping a clean gold electrode in 1 mM mercaptopropionic acid in ethanol for 12 h at room temperature. The electrode was thoroughly rinsed with ethanol to remove the physically adsorbed thiol and dried. Then the electrode was placed into AuNPPEG solution for 4 h to form Au/MPA/AuNPPEG electrode. The Au/MPA/AuNPPEG electrode was dipped in 1 mM DNA-c probe solution in conjugation buffer for overnight at 4 °C. Then, the electrode was washed with 10 mM PBS solution to remove unbounded DNA strands. This electrode was used for the detection of complementary DNA throughout the experiment. The obtained electrode was denoted as Au/MPA/AuNPPEG /DNA-c electrode. The Au/MPA/AuNPPEG/DNA-c electrode was immersed into a solution containing various concentration of target DNA (DNA-t) in 0.3 M PBS (pH 7) at 25 °C for 2 h. The complementary portions of the DNA-t hybridized with the immobilized DNA-c. Subsequently, the electrode was dipped in the buffer solution to remove nonspecifically adsorbed DNA-t, and then washed and dried. The hybridization with DNA-r.AuNPMPA was done with same hybridization procedure. Then the Au/MPA/AuNPPEG/DNA-c|DNAt|DNA-r.AuNPMPA electrode was transferred into the electrochemical cell system containing 10 mM PBS for CV and chronoamperometry measurements. To study the effect of labeled gold nanoparticles in the signal enhancement, we conducted the same experiments without AuNPMPA. 2.6. Regeneration of sensor surface For regeneration of the sensor surface, the electrodes obtained after the complete DNA hybridization (Au/MPA/AuNPPEG /DNA-c|DNA-t|DNA-r.AuNPMPA electrode) was immersed in 0.2 M NaCl solution for 10 min followed by washing with 10 mM PBS solution containing 1% SDS (Ensafi et al., 2011; Chu et al., 2008). The dehybridization of DNA is due to the chaotropic action of SDS on biomolecules. Regeneration of the sensor surface was monitored using chronoamperometry and the reusability of the biosensor surface was tested by repetitive hybridization with

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different target concentrations. The stability of the sensor was also investigated after 15 days by keeping at 4 °C.

3. Results and discussion 3.1. Sensor fabrication The schematic representations of the various stages of sensor fabrication and DNA hybridization related to specific BRCA1 sequences are shown in Scheme 1. The sensor was developed by the layer-by-layer assembly of mercaptopropionic acid (MPA), polyethylene glycol (PEG) functionalized gold nanoparticle (AuNPPEG), capture DNA (DNA-c), target BRCA1 DNA (DNA-t) and gold nanoparticle labeled reporter DNA (DNA-r.AuNPMPA) on gold electrode. The MPA monolayer on the electrode surface prevents the non-specific adsorption of DNAs during immobilization and hybridization. To this MPA monolayer AuNPPEG was immobilized through hydrogen bonding. Functionalized gold nanoparticles on the MPA monolayer leads to efficient immobilization of single stranded DNA-c through hydrogen bonding between the PEG and cytosine and it nullifies the insulating effect of MPA monolayer by its electron conducting property. The hydrogen bond formed between functionalized gold nanoparticles and single stranded DNAs helps to effectively immobilize the DNAs on the sensor surface. The washing procedure in each step will not affect this bonding. Hybridization of target probe and reporter probe DNAs were done in the traditional sandwich detection strategy. One half of the DNA-t was allowed to hybridize to DNA-c using the hybridization buffer. After washing the electrode, DNA-r.AuNPMPA was allowed to hybridize to the second half of the DNA-t. 3.2. Characterization of sensor surface 3.2.1. Cyclic voltammetry Cyclic voltammetry was conducted in 5 mM K3[Fe(CN)6] /0.1 M KCl and in 10 mM PBS buffer (pH 7) at 0.1 V/s using modified electrodes. Fig. 1 compares the voltammetric response at each stage of the modification process. Self-assembly of MPA generated an insulating layer on the electrode surface leading to a reduction in current value. The current obtained after AuNPPEG immobilization showed an increase from the Au/MPA surface. The immobilization of DNA-c and hybridization of DNA-t did not produce any noticeable difference in the current value. But after the hybridization with DNA-r.AuNPMPA, it showed a remarkable

Scheme 1. The schematic representations of the various stages of sensor fabrication and DNA hybridization related to specific BRCA1 sequences. (a) Au (b) Au/MPA (c) Au/MPA/AuNPPEG (d) Au/MPA/AuNPPEG/DNA-c (e) Au/MPA/AuNPPEG/DNA-c|DNA-t and (f) Au/MPA/AuNPPEG/DNA-c|DNA-t|DNA-r.AuNPMPA.

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50 aM of DNA-t. When the target concentration was less than 50 aM, the hybridization signal remained almost the same as that of background surface (Au/MPA/ AuNPPEG/DNA-c). The current response of the sensor is found to be linear with logarithmic concentration of DNA-t from 50 aM to 1 nM (Fig. 3(B)). The electrochemical impedance spectroscopy (EIS) analysis of the developed sensor with different DNA-t concentrations was also performed. The Nyquist plots of electrochemical impedance spectroscopy with different DNA-t concentrations are given in Fig. 3(C) which showed a continuous decline in the charge transfer resistance. The DNA-t concentration was varied from 100 aM to 1 nM and it was seen that the resistance decreased with increase in DNA-t concentration due to the presence of more number of gold nanoparticles on the surface with increase in DNA-t concentrations.

Fig. 1. Cyclic voltammograms of the sensor during each stages of modifications. (a) Au (b) Au/MPA (c) Au/MPA/AuNPPEG (d) Au/MPA/AuNPPEG/DNA-c (e) Au/MPA/AuNPPEG /DNA-c|DNA-t (f) Au/MPA/AuNPPEG/DNA-c|DNA-t|DNA-r.AuNPMPA. The electrochemical measurements were carried out in 5 mM K3[Fe(CN)6] and 0.1 M KCl at a scan rate of 0.1 V/s.

increase in current value due to the presence of gold nanoparticles on the surface. 3.2.2. Morphological analysis The modification of Au electrode at each stage was also monitored by scanning tunneling microscopy, scanning electron microscopy and atomic force microscopy. Fig. 2 shows the STM current images of the electrode at different modification steps. The bare Au surface showed a current value of nano ampere and it decreased to pico ampere after the formation of self-assembled monolayers of MPA. The immobilization of DNA-c, hybridization of DNA-t and DNA-r.AuNPMPA are also clearly visible in the STM images. The SEM images of the electrode at different modification steps are shown in Fig. S1. Significant morphological changes were observed for the surface during each step of modification. The bare Au surface was flat with some roughness and Au/MPA surface was smooth due to the formation of self-assembled monolayers of MPA. The presence of AuNPPEG was clearly seen in the SEM image of the Au/MPA/AuNPPEG surface. The immobilization of DNA-c and hybridization of DNA-t gives some smoothness to the surface. After the hybridization with DNA-r.AuNPMPA, an increase in the number of gold particles on the surface was observed confirming the presence of DNA-r.AuNPMPA. From the SEM images, it was found that the average diameter of gold nanoparticles on the surface is 20–25 nm. The AFM images of the sensor at each modification steps are shown in Fig. S2. The AFM images are also in accordance with the SEM images. Gold nanoparticles can be seen in AFM images after the immobilization of AuNPPEG. 3.3. Detection of target DNA 3.3.1. Electrochemical detection: chronoamperometry and electrochemical impedance spectroscopy Chronoamperometry was used to measure the changes in current with respect to time at an applied potential of  0.35 V during the hybridization of various concentrations of DNA-t with DNA-r.AuNP (10 nM DNA-r concentration). The potential of 0.35 V was randomly selected because the cyclic voltammograms in 10 mM PBS buffer does not show any specific peak (fig.S3). Specific hybridization signals were observed over the range of 50 aM to 1 nM target DNA concentration (Fig. 3(A)). It was seen that the increase in current is proportional to the increase in concentration of DNA-t and the change was detectable up to

3.3.2. Scanning tunnelling spectroscopy The STS analysis of the developed sensor with different DNA-t concentrations was used to support the detection capability of the modified sandwich type sensing mechanism. The STS curves with DNA-t concentrations from 100 am to 100 pM are given in Fig. 4 (a). The current increases with increasing concentration of DNA-t which supports the chronoamperometry and EIS results. 3.3.3. EQCM measurements EQCM experiments were carried out to support the detection capability of this sensor. The same sandwich type detection method shown in Scheme 1 was adopted in EQCM experiment except that the gold polished quartz crystal electrode was used instead of polycrystalline gold electrode. The frequency of the crystal was measured before and after the hybridization of DNA-r.AuNPMPA and plotted with respect to the logarithmic concentration of DNA-t in Fig. 4(b). It is evident from the figure that the detection limit of the sensor is 100 aM and the frequency change of the sensor surface is linear to the logarithmic concentration of DNA-t from 100 aM to 1 nM. EQCM was further used to calculate the number of molecules on the surface (see below section 3.6). 3.4. Regeneration of sensor surface The regeneration of sensor surface was achieved by immersing the hybridized electrodes in 0.2 M NaCl solution for 10 min followed by washing with 10 mM PBS solution containing 1% SDS. The sensor surface could be regenerated by 96.66 7 0.95% approximately. The chronoamperometric response showing the regeneration of sensor surface is given in Fig. 5(a). The regeneration procedure allows the hybridized DNA strands to be dehybridized and it does not affect the binding between the functionalized gold nanoparticle and DNAs. This can be explained as the difference in the mechanism of action of SDS on DNA and on carboxylic acid. It may be possible that in presence of NaCl the Na þ shields the phosphate groups of DNA and due to the presence of anionic groups in SDS, it interact with the excess Na þ and alter the hydrophobic effect in the DNA structure and decreases the stability of DNA helix. Thus after regeneration DNA-r.AuNP and DNA-t are removed from the surface leaving behind the DNA-c. It is confirmed that the amperometric signal of the background surface (Au/MPA/AuNPPEG/DNA-c) before and after regeneration was approximately the same. Regeneration was again confirmed from the frequency change measurement from the EQCM which showed that the frequency of the regenerated surface retains its value of the background surface (Au/MPA/AuNPPEG/DNA-c). The regenerated surface was used for further hybridization with target DNA. It was noted that the same current has been obtained upon using

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Fig. 2. STM images of the sensor surface during different stages of modifications. (a) Au, (b) Au/MPA, (c) Au/MPA/AuNPPEG, (d) Au/MPA/AuNPPEG /DNA-c, (e) Au/MPA/AuNPPEG /DNA-c|DNA-t and (f) Au/MPA/AuNPPEG /DNA-c|DNA-t|DNA-r.AuNPMPA.

the same concentration of target DNA for hybridization before and after the dehybridization step. 3.5. Selectivity and stability of the sensor surface Selectivity of the sensor was investigated by hybridization experiments with non complementary (NC) DNA-t and 3 base mismatch (3MM) DNA-t sequences. 1 pM of DNA-t, NC DNA-t and 3MM DNA-t were used for the experiment. The hybridization signals were monitored using chronoamperometry and STS.

In hybridization experiments with non complementary sequences, no or small change in current was observed from the background surface in the chronoamperometric measurements. The current value was increased with 3 base mismatch complementary sequences and complementary sequences. The increase was much higher in complementary sequence than the three base mismatch. This shows that the proposed sensor is selective to complementary sequences and it can be used to detect mismatch sequences. The comparison of the experiments with complementary target, non complementary target and 3 base mismatch complementary

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Fig. 3. (A) Chronoamperometric response of the sensor on hybridization of DNA-r.AuNPMPA with various DNA-t concentrations on the surface. Background corresponds to Au/MPA/ AuNPPEG /DNA-c surface. (B) Current versus log molar concentration plot of the sensor from 50 aM to 1 nM DNA-t concentration. (C) The Nyquist plots of electrochemical impedance spectroscopy (EIS) with different DNA-t concentrations a)100 aM, b) 1 fM, c)1 pM, d) 100 pM and e) 1 nM.

Fig. 4. (a) The STS curves with different DNA-t concentrations. The experiments were performed in the range of  1.5 to þ 1.5 V with set points 0.5 nA and 0.1 V at 25 °C, 40– 50% humidity. (b) Frequency change (upon hybridization with DNA-r.AuNPMPA) with logarithmic concentration of DNA-t. The error bar gives the standard deviation of three repetitive measurements.

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Fig. 5. (a) Chronoamperometric response showing the regeneration of sensor surface. (b) Bar diagram showing the selectivity of the sensor surface by chronoamperometry measurements. (c) Bar diagram showing the selectivity of the sensor surface by STS curve measurements.

sequences are shown in Fig. 5(b). Moreover, we noted that the three base mismatch induced much smaller changes in the current than the complementary target at all concentrations. The slope of the calibration curve for complementary DNA was  1.3 times higher than that of the slope recorded for the three base mismatch (Fig. S4 (b)). STS indicate a significant variation in the current upon hybridization with complementary, 3 base mismatch and non complementary sequences. The selectivity as observed in STS is given in Fig. 5c. The experimental curves are given in supplementary information (Fig. S4). The stability of the sensor was also studied. The electrodes were stored at 4 °C. The result showed that 92% of signal was retained after two weeks (data not shown) and the sensor is stable. 3.6. Effect of AuNPMPA and AuNPPEG The effect of AuNPMPA in the signal enhancement was studied by comparing the hybridization of DNA-t (1 pM concentration) with DNA-r alone and with DNA-r.AuNPMPA (Fig. S5 (a)). A higher current was observed in DNA-r.AuNPMPA which suggests that the sensitivity observed in the developed sensor is greatly due to the presence of AuNPMPA. We have carried out the experiments with AuNPPEG as a label for DNA-r instead of AuNPMPA. The results showed that the sensor with labeled AuNPPEG showed a lesser current than AuNPMPA which is attributed to the effect of lower chain length of the MPA compared to the PEG which allowed

efficient electron transfer. However, the use of AuNPPEG in the first step is justified by the fact that a higher loading of DNA-c strands is possible with the PEG spacer (Hurst et al., 2006). To investigate the effect of AuNPPEG in the sensitivity of the sensor, we have carried out experiments with citrate capped gold nanoparticles (AuNPcitrate) instead of AuNPPEG. A higher current is observed in the AuNPPEG is due to the higher loading of DNA-c on AuNPPEG. The chronoamperometric response of the experiments with AuNPcitrate and AuNPPEG are given in Fig. S5 (b). To verify the higher loading of DNA-c on the AuNPPEG, we measured the change in frequency of EQCM during the immobilization of DNA-c on AuNPPEG and AuNPcitratre. From the change in frequency, adsorbed mass and the number of DNA-c immobilized on the quartz crystal with AuNPcitrate and AuNPPEG was calculated. It was found that 2.737  1013 molecules/cm2 of DNA-c was immobilized on AuNPPEG compared with 1.6422  1013 molecules/cm2 of DNA-c immobilized on AuNPcitratre (see supporting information).

4. Conclusion An attomolar sensitive genosensor has been developed for the detection of BRCA1 gene sequences. The sensor was evaluated using chronoamperometry, EIS and STS. The detection limit of the sensor was found to be 50 attomolar BRCA1 gene and the sensor exhibited excellent selectivity against non complementary

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sequences and three base mismatch complementary sequences. It was evident from the experiments that the higher sensitivity of the proposed sensor is greatly due to the high electron conduction through the use of two appropriately functionalized gold nanoparticles. This DNA sensor showed good reproducibility, stability and reusability. EQCM experiments were carried out to support the detection capability of the sensor using gold polished quartz crystal electrode. This sensor could prove its potential application in cancer biomarker detection in the early stages of cancer, where the concentration of biomarkers is very low.

Acknowledgements The authors acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India and National Institute of Technology Calicut for providing the facility and financial support. We thank Dr. Abraham Mathew and Mr. Mathew Kuruvilla, Department of chemistry, Calicut University for the help they offered for the EIS measurements.

Appendix A. Suplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.054.

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Attomolar detection of BRCA1 gene based on gold nanoparticle assisted signal amplification.

In this work, we report a simple strategy for signal amplification using appropriately functionalized gold nanoparticles in an electrochemical genosen...
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