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A highly sensitive DNA sensor for attomolar detection of the BRCA1 gene: signal amplification with gold nanoparticle clusters P. Abdul Rasheed and N. Sandhyarani*

Received 2nd January 2015, Accepted 3rd February 2015

Single stranded DNA fragments were conjugated onto gold nanoparticles leading to the formation of gold

DOI: 10.1039/c5an00004a

implemented for signal amplification in an electrochemical DNA sensor based on a graphene substrate. The sensor exhibited excellent sensitivity and selectivity with a detection limit of 50 attomolar target DNA.

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nanoparticle clusters upon hybridization with complementary strands. These clusters were successfully

Introduction The development of very sensitive and fast analytical techniques is of high priority due to new challenges faced by society, particularly the fast spreading of infectious diseases. Sequencespecific detection of nucleic acid is essential for the detection of clinical pathogens and early stage diagnosis of cancer. Among a wide variety of analytical techniques, electrochemical methods of detection have been very promising as they can be applied using very simple instrumentation, are cost effective and can give a quick and sensitive response.1–3 In general, electrochemical DNA sensors monitor the formation of a DNA duplex (by means of the hybridization of the target DNA to the immobilized capture ssDNA) through changes in current or potential values either using electrochemical labels or label free systems.4–7 The most important process in the fabrication of an electrochemical DNA sensor is the preparation of a well defined DNA recognition interface. The effective immobilization of capture probe DNA onto the transducer surface through a suitable matrix and the immobilization method used are crucial in the development of sensors. Various electrode materials such as gold, carbon nanotubes (CNTs), graphene coated electrodes and modified glassy carbon electrodes (GCEs) have been used for DNA immobilization.8–11 Graphene is widely used as a platform for biomolecule immobilization due to its large surface area to volume ratio, high conductivity and electron mobility at room temperature, and its biocompatibility.12,13 Non-covalent interactions such as π–π stacking or hydrogen bonding between graphene and biomolecules make graphene a promising candidate for conjugation with various biomolecules and for application

Nanoscience Research Laboratory, School of Nano Science and Technology, National Institute of Technology Calicut, Calicut, Kerala, India. E-mail: [email protected]; Fax: +91 495 2287250; Tel: +91 495 2286537

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in sensors. Single stranded DNAs (ssDNAs) are directly immobilized on graphene and graphene oxide transducers through π electron interactions.14–17 Normally, when a redox label such as ferrocene or methylene blue is used for the electrochemical detection of DNA, it is attached to the unbound end of DNA. Recently, the introduction of nanomaterials in biosensor design has greatly improved the analytical performance of the sensors. 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.18–21 One of the most accepted methods for the detection of DNA is based on the sandwich assay. In this assay, a surface bound capture probe DNA (DNA-c) specifically hybridizes to one specific region of target DNA (DNA-t). To complete the assay, a second probe called the reporter probe (DNA-r) usually labelled with an electroactive moiety is hybridized to the unhybridized region of the target DNA forming a sandwich of DNA-c|DNA-t|DNA-r.22 Despite its advantages, one of the major limitations of the sandwich assay is that the DNA-t hybridizes to a single DNA-c and DNA-r limiting the signal amplitude and sensitivity. A simple and reliable approach to improve the sensitivity could augment the utility of such sensors. The use of gold nanoparticle clusters has been reported in different sensing applications. The majority of these sensors are based on surface enhanced Raman spectroscopy,23–25 fluorescence26,27 and colorimetric methods.28–30 Xia et al. reported a label-free and sensitive strategy for the detection of microRNAs based on the difference in the structure of RNA versus DNA and the dual-amplification of 4-mercaptophenylboronic acid (MBA) capped AuNPs (MBA-AuNPs) and electrochemically active dopamine (DA)-capped AuNPs (DA-AuNPs).31 The amplified electrochemical sensing of DNA by the analyte-induced aggregation of nucleic acid-functionalized Au nanoparticles was reported.32 A gold nanorod assembly based immunosensor

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has been fabricated for the detection of α-fetoprotein which uses differential pulse voltammetry (DPV) for the detection.33 Here the host–guest assembly was used. Recently, an impressive number of reports related to a DNA sensor for BRCA1 gene detection using different nanomaterials as well as different transduction mechanisms have appeared.34–40 In this work, we are exploring the use of clusters of gold nanoparticles which are formed by the self-assembly of ssDNA stabilized gold nanoparticles (AuNP) as an electrochemical probe for the sensitive detection of the BRCA 1 gene (Scheme 1). To the best of our knowledge, this is the first report in which gold nanoparticle clusters are used as an electrochemical probe leading to enhancement in the electrochemical detection of the BRCA1 gene. Here, the capture probe DNA (DNA-c) with an amino group at the 5′ end was immobilized on a graphene modified glassy carbon electrode (GCE) through π–π stacking interactions.14 The reporter probe DNA is conjugated to a cluster of gold nanoparticles. Sequences for the reporter and capture DNAs were selected such that one half of the BRCA1 gene hybridizes to the immobilized DNA-c and the other half to the reporter probe DNA (DNA-r) conjugated to a AuNP cluster. In this work, the DNA-r conjugated to a AuNP cluster is referred to as DNA-r·AuNPC. The generation of the analytical signal was by the electrochemical oxidation of the gold nanoparticles in the presence of HClO4 which was monitored using cyclic voltammetry/chronoamperometry to detect the concentration of BRCA1 gene. The sensitivity of this sensor towards target DNA is in the attomolar range, offering orders of magnitude lower detection limit than most of the reports (see below in Table 1).

Experimental methods Materials 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′ Complementary reporter probe (cDNA-r): 5′ GAA GAA AAT C 3′ Non complementary probe (NC): 5′ CCT TGT TGG ACT CCC TTC T 3′ Three base mismatch complementary probe (3MM): 5′ C̲AA CAA AAG C̲AA C̲AA AAT C 3′ Graphite powder (>20 μm), O-(3-carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]-polyethylene glycol (CPEG), N-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 3-sulfo-N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich India. Chloroauric acid was purchased from SRL chemicals, India. Other chemicals used were of analytical reagent grade and they were supplied from Fischer Scientific and Merck, India. Ultrapure and deionized water was used in all experiments. The conjugation buffer and hybridization buffer used were 0.1 M NaCl PBS buffer and 0.3 M NaCl PBS buffer, respectively.41,42 The 0.1 M NaCl PBS buffer consists of 0.1 M NaCl in 10 mM phosphate buffer (pH 7) and the 0.3 M NaCl PBS buffer consists of 0.3 M NaCl in 10 mM phosphate buffer (pH 7). The glassy carbon electrode was used as the sensor surface for electrochemical measurements. Instruments Cyclic voltammetric (CV) and chronoamperometry measurements were performed with a CHI 400A electrochemical analyzer (CH Instruments, Texas, USA). A three-electrode system was employed with Pt wire as the auxiliary electrode, calomel electrode as the reference electrode, and modified glassy carbon electrode (GCE) as the working electrode. Electrochemical measurements were carried out at room temperature with a scan rate of 0.1 V s−1. Scanning electron microscopy (SEM) images were taken using an SU6600 variable pressure field emission scanning electron microscope (Hitachi, Japan). Atomic force microscopy (AFM) images were taken using an XE-100 Atomic force microscope, Park systems, Korea.

Scheme 1 A cartoon representation of the BRCA1 sensor and the DNA sequences used for the sensor fabrication. The unhybridized DNA-r on the outer gold nanoparticles of the cluster undergoes hybridization with DNA-t.

Table 1

Synthesis of graphene Graphene was synthesized by a reported procedure. The synthesized graphene was found to be a few layers thick.43 The

DNA sensors with AuNP assembly: a comparison

Author

Cluster used

Techniques used

Detection limit

Ref.

L. Guo et al. (2013) Y. Zhang et al. (2012) X. Gu et al. (2014) S. Dinda et al. (2013) P. Yuan et al. (2012) This work

Target induced AuNP dimer DNA-AuNP clusters Magnetic hybrid AuNPs cluster conjugate Self-assembled gold nanoparticle cluster arrays dsDNA coupled Au NP assemblies DNA-AuNP clusters

Colorimetric Colorimetric Fluorescence SERS Photoluminescence Chronoamperometry

1 pM 1 nM — 10 nM 2.9 pM 50 aM

28 30 26 25 47 —

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graphene was dispersed in a dimethyl formamide (DMF)–water mixture (1 : 9 ratio) with a concentration of 0.5 mg ml−1 and used for further experiments.

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Synthesis of DNA-r·AuNPC The gold nanoparticles were synthesized by monosodium glutamate reduction as described in the literature.44 These gold nanoparticles were functionalized with the DNA reporter probe as described in our earlier work and it is denoted as DNAr·AuNP.45 Using the same procedure, AuNPs stabilized with complementary strands of the DNA-r (i.e. cDNA-r) also were synthesized and referred to as cDNA-r·AuNP. AuNPCs are synthesized by mixing the AuNPs conjugated with DNA-r (DNAr·AuNP) and the AuNPs conjugated with complementary strands of DNA-r (cDNA-r·AuNP). The concentrations of DNA-r and cDNA-r were selected such that there were equal numbers of DNA strands attached onto the gold nanoparticles in both DNA-r·AuNP and cDNA-r·AuNP. The concentration of DNAr·AuNP in the mixture was higher than the concentration of cDNA-r·AuNP, in order to form the AuNPC with cDNA-r·AuNP in the centre surrounded by DNA-r·AuNPs. The cDNA-r·AuNPs and DNA-r·AuNPs were mixed in the ratio 1 : 4 in the presence of hybridization buffer for 4 hours at 37 °C to form DNA-r·AuNPC. The unreacted reagents were removed by centrifugation at 12 000 rpm for 30 minutes and the clusters were purified by repeated centrifugation. We noted that a mixture of clusters was formed in this procedure. The separation of DNA-r·AuNPC was done by repeated centrifugation at different speeds. Initially, the precipitate was dispersed in buffer and centrifuged at 10 000 rpm for 10 minutes. The precipitate was collected, redispersed in buffer and centrifuged at 5000 rpm for 10 minutes and the supernatant was taken. The supernatant was analyzed by SEM and it was found that these are clusters of AuNPs with one nanoparticle in the centre which is surrounded by 5–6 gold nanoparticles with hybridized double stranded DNAs. These were used in all the electrochemistry experiments. The synthesized clusters were stored at 4 °C when not in use.

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dropped onto the surface for 1 h at room temperature and then the electrode surface was washed. The resulting GCE/Gr/ DNA-c|DNA-t |DNA-r·AuNPC was used for the electrochemical measurements. A 0.2 M perchloric acid (HOCl4) solution was used as the electrolyte.

Results and discussion The schematic illustration of the BRCA1 sensor and DNA sequences used for the sensor fabrication is given in Scheme 1. Synthesis of DNA-r·AuNPC The number of nanoparticles in the cluster was controlled by the relative concentration of cDNA-r·AuNP and DNA-r·AuNP. The central particle in the structure is connected to each peripheral one via a double-stranded DNA linker and each of the peripheral AuNPs contain unhybridized ssDNA-r. The SEM and AFM images of DNA-r·AuNPC with 1 : 4 ratios of cDNAr·AuNP and DNA-r·AuNP are given in Fig. 1. The images confirmed that one gold nanoparticle was surrounded by 5–6 gold nanoparticles to form the AuNPC. The SEM images of the AuNP clusters with different ratios (1 : 1 and 1 : 7) of DNAr·AuNPs and cDNA-r·AuNPs are given in Fig. 2. It is seen that on using a 1 : 4 ratio, the cluster is more uniform and this cluster was used for the sensor fabrication. Characterization of sensor surface The SEM images of the sensor surface before and after hybridization with DNA-r·AuNPC are shown in Fig. 3. Indium tin oxide (ITO) coated glass was used as the substrate for SEM analysis. The presence of gold nanoparticle clusters on the surface is clearly evident in the SEM images after hybridization with DNA-r·AuNPC. When the non-complementary sequence of

Sensor fabrication Prior to modification, the working GCE was polished with 0.3 µm alumina slurry and washed thoroughly with water. The freshly polished electrodes were then pretreated by cleaning them with isopropanol followed by sonication in water. A 6 μl solution of graphene was dropped onto the cleaned GCE and allowed to dry in ambient air for 2 h to obtain graphene modified GCE. 6 μl of 1 μM DNA-c solution in conjugation buffer was dropped onto the graphene modified GCE and maintained at 4 °C for 2 h. The ss-DNA is conjugated on the graphene layer through π–π stacking interactions.14 Nonspecific binding of single stranded DNAs was blocked by dropping 1 M bovine serum albumin (BSA) for 1 min onto the surface. Then DNA-t solution in hybridization buffer was dropped onto the surface and kept there for 1 h at room temperature. Half of the DNA-t gets hybridized with DNA-c. The surface was washed and finally, the DNA-r·AuNPC solution in hybridization buffer was

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Fig. 1 (a) SEM image of DNA-r·AuNPC. (b) AFM 2D image and (c) AFM 3D image of DNA-r·AuNPC.

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Fig. 2 SEM images of the AuNP clusters with different ratios of DNA-r·AuNPs and cDNA-r·AuNPs. (a) 1 : 1 and (b) 1 : 7.

Fig. 3 SEM images of (a) ITO/graphene/DNA-c|DNA-t. (b) ITO/graphene/DNA-c|DNA-t|DNA-r·AuNPC with 1 pM DNA-t concentration, (c) 1 nM DNA-t concentration and (d) 1 μM DNA-t concentration.

DNA-t was used for hybridization under the same conditions the gold nanoparticle clusters were not observed as expected, which confirmed that there is no nonspecific binding of DNA on the surface. These results confirm the successful immobilization of DNA-c, its hybridization with DNA-t and the hybridization of DNA-t with DNA-r·AuNPC. Concentration dependence has also been observed on the surface. The number of gold nanoparticle clusters increases with the increase in concentration of DNA-t. However, it is seen that even after using a higher concentration of DNA also, not all regions of the graphene are covered uniformly. This indicates that not all capture DNAs are oriented perpendicular to the surface. Some may be parallel to the graphene surface due to the π–π stacking, and therefore do not undergo hybridization with the DNA-t. Taking this fact into account, we have used an excess of capture DNA compared to the target DNA for efficient hybridization. Electrochemical analysis To validate the enhanced sensitivity in the detection of the BRCA1 gene using the AuNPC modified DNA-r, we measured the cyclic voltammograms of the sensor in the presence of

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Fig. 4 Cyclic voltammograms of the GCE/graphene/DNA-c|DNA-t electrode with (a) 1 nM of DNA-t on hybridization with DNA-r, DNA-r AuNP and DNA-r·AuNPC and (b) at different DNA-t concentrations on hybridization with DNA-r·AuNPC. The electrochemical measurements were carried out in an aqueous solution of 0.2 M perchloric acid (HClO4) vs. SCE at a scan rate of 0.1 V s−1.

the DNA-r·AuNPC, in the presence of DNA-r·AuNP and in the presence of DNA-r without AuNPs in 0.2 M perchloric acid (HClO4). A strong oxidation peak current due to the oxidation of the gold nanoparticles was observed at 1.1 V in the presence of AuNPC, however the peak current was lower in the presence of AuNPs. Obviously the peak was not observed when the DNA-r without gold nanoparticles was used for hybridization (Fig. 4(a)). The presence of the oxidation peak at 1.1 V indicates the hybridization of DNA-t with DNA-c and further hybridization of DNA-r·AuNPC with DNA-t, leading to a long range electron transfer from the AuNPs through the DNA duplex to the graphene. This signal was used as the sensing signal in CV.46 The signal amplification of the AuNPC based assay leads to a significant improvement in the detection limit, and we achieved a detection limit of 100 aM using cyclic voltammetry. Using AuNPs the detection limit was 1 fM.39 The performance of the graphene-DNA sensor electrode for concentration dependent sequence-specific detection is given in Fig. 4(b). In this experiment, the concentration of DNA-t on the GCE/Gr/DNA-c|DNA-t| DNA-r·AuNPC electrode was varied from 100 aM to 1 nM. A higher concentration of DNA-r·AuNPC (approx. 10 nM DNA-r concentration) was used in each experiment to guarantee the complete hybridization to the DNA-t, and after hybridization the unhybridized DNA-r·AuNPC was washed out prior to the measurement. The number of gold nanoparticles on the surface increases with the increasing concentration of DNA-t as it allows more DNA-r·AuNPC to hybridize. Hence the peak due to gold nanoparticle oxidation increases with the increasing concentration of DNA-t, leading to the sensitive detection of DNA-t. The addition of non-complementary DNA-t sequences under the same conditions did not show the oxidation peak as there was no hybridization of DNA-t with DNA-c. Chronoamperometric responses of GCE/Gr/DNA-c|DNA-t| DNA-r·AuNPC with varying concentrations of DNA-t were monitored at 1.1 V and this signal was used as the sensing signal in chronoamperometry. A monotonic increase in the redox current was seen until a signal gain of 250% was achieved with 1 nM DNA-t (Fig. 5(a)). A detection limit of 50 aM is achieved using chronoamperometry (signal with a clear difference from blank). However, the cyclic voltammetry analysis did not give

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Conclusion In summary, we demonstrated that the incorporation of AuNP clusters as a label for reporter probe DNA leads to highly sensitive electrochemical detection of target DNA. The sensor is of low cost and is a disposable one which displayed very good selectivity and sensitivity. The sensor could detect up to 50 attomolar target DNA. Given that 6 μl DNA-t was used in this experiment, which amounts to approximately 180 molecules of DNA-t on the surface, the sensitivity achieved is very high. The sensor exhibited good selectivity against non-complementary sequences and three base mismatch complementary targets. The developed sensor surface could find applications in early cancer diagnosis owing to its high sensitivity and selectivity.

Fig. 5 (a) Chronoamperometric response of the sensor with various DNA-t concentrations. (b) Dependence of the redox current at the oxidation potential (1.1 V) on the DNA-t concentration. The redox current with DNA-r·AuNP as the label is given for comparison. (c) Chronoamperometry response showing the selectivity of the sensor with 1 pM concentration of DNA-t.

any peak for a 50 aM concentration. So we have taken 100 aM to be the detection limit of the developed sensor. The redox current vs. log of the concentration of DNA-t follows a linear trend from 100 attomolar to 1 nanomolar (Fig. 5(b)). The current at t = 1 s was used for plotting the current vs. log molar concentration curve. Our previous work with gold nanoparticles used as a label instead of gold nanoparticle clusters provides the detectable change for 1 fM of DNA-t39 and it is shown that the use of gold nanoparticle clusters enhances the detection capability due to the increased number of gold nanoparticles on the surface. The selectivity of the sensor was studied by measuring the chronoamperometry response with non-complementary DNA-t and three base mismatch target DNA. The chronoamperometric responses were measured and it is plotted as the current change from the background surface (Fig. 5(c)). From the results, it is found that the sensor is selective to sequences. The selectivity can be further improved by using specific redox mediators such as methylene blue or ruthenium complexes. Table 1 tabulates a comparison of reported sensors using AuNP clusters or assemblies. We noted that the regeneration of the sensor surface is not effective and hence the developed sensor can be used for a one time measurement. To authenticate the performance on a disposable sensor we used a screen printed electrode (SPE) and the chronoamperometric responses were measured. The screen printed electrodes were fabricated with a carbon paste working electrode, a carbon paste counter electrode and Ag/ AgCl reference electrodes. The working electrode was modified using the same strategy as for the GCE. The results confirmed that the SPE/Gr/DNA-c|DNA-t|DNA-r·AuNPC is highly sensitive and the results with SPE are comparable to those obtained with the modified GCE.

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Acknowledgements This work was supported by the Council of Scientific and Industrial Research, INDIA (no. 01/(2762)/13/EMR-II). Financial support by Department of Science and Technology, Government of India also is acknowledged.

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A highly sensitive DNA sensor for attomolar detection of the BRCA1 gene: signal amplification with gold nanoparticle clusters.

Single stranded DNA fragments were conjugated onto gold nanoparticles leading to the formation of gold nanoparticle clusters upon hybridization with c...
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