Signal amplification for impedimetric genosensing using gold-streptavidin nanoparticles

Available online at www.sciencedirect.com

Electrochimica Acta 53 (2008) 4022–4029

Signal amplification for impedimetric genosensing using gold-streptavidin nanoparticles A. Bonanni, M.J. Esplandiu, M. del Valle ∗ Sensors and Biosensors Group, Department of Chemistry, Universitat Aut`onoma de Barcelona, Edifici Cn, 08193 Bellaterra, Barcelona, Spain Received 31 May 2007; received in revised form 5 November 2007; accepted 12 November 2007 Available online 19 November 2007

Abstract Streptavidin-coated gold nanoparticles (strept-AuNPs) were used in this work to amplify the impedimetric signal generated in a biosensor detecting the DNA hybridization event. Probe oligomer was adsorbed onto a graphite epoxy composite (GEC) electrode surface and the impedance measurement was performed in a solution containing the redox marker ferrocyanide/ferricyanide. The biotinylated complementary oligomer was used as target. The change of interfacial charge transfer resistance (Rct ), experimented by the redox marker, was recorded to confirm the hybrid formation. The addition of strept-AuNPs, binding to the target due to the strong streptavidin–biotin interaction, led to a further increment of Rct thus obtaining significant signal amplification. Strept-AuNPs on the electrode surface were observed by scanning electron microscopy (SEM) after silver enhancement treatment. A competitive binding assay was also performed using unlabelled DNA target to demonstrate its applicability to real sample analysis. © 2007 Elsevier Ltd. All rights reserved. Keywords: DNA; Impedance; Streptavidin; Nanoparticles; Biosensor

1. Introduction The development of DNA biosensors (genosensors), has been offering a valid alternative to the more classical methods for DNA analysis. In fact, these devices present several advantages in comparison with traditional approaches, such as low cost, rapid analysis, possibility of miniaturization and analysis in situ [1,2]. Genosensors can be classified, depending on the technique employed for the transduction, in optical [3], piezoelectric [4] or electrochemical [5]. Recently, among available electrochemical techniques [6–12], electrochemical impedance spectroscopy (EIS) [13,14] is rapidly developing as a tool for studying DNA hybridization [15,16]. This is due to its ability to directly probe the interfacial properties (capacitance, charge transfer resistance) of modified electrodes [17–24]. Different protocols have been proposed for DNA analysis using EIS. Some of them are based on the direct measurement of capacitance [25–27] whilst others employ a redox marker to detect

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Corresponding author. Tel.: +34 935811017; fax: +34 935812379. E-mail address: [email protected] (M. del Valle).

0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.11.030

the hybridization signal [28,29]. Simultaneously, protocols have been modified trying to amplify the impedimetric signal. For this reason, procedures based on enzyme schemes [30,31], DNA modification with biomolecules [32], or the use of nanoparticles [28,33,34] have been proposed. Metal nanoparticles offer new possibilities for the progress of both chemical and biological sensing [35]. From the variants gathered from the literature, some employ oligonucleotides modified with metal nanoparticles to enhance the impedimetric signal [28], others use electrodes modified with gold nanoparticles in order to increase the amount of DNA adsorbed onto the electrode surface [36,37], others exploited a dendritic signal amplification of oligonucleotide-functionalized metal nanoparticles [38]; some others utilized gold nanoparticle labels for electrochemical detection of DNA hybridization by stripping [39,40]. Silver enhancement treatment, i.e. silver deposition on gold nanoparticles, is generally used to visualize antibody, protein or DNA conjugated particles both in electron microscopy studies [41,42] and electrochemical detection [43], i.e. determination of silver by differential pulse voltammetry (DPV) [44]. In this work, the use of strept-AuNPs is proposed for the amplification of the impedimetric signal generated by

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DNA hybridization. The transducer employed consisted of a graphite–epoxy composite (GEC) electrode, of general use in our laboratories and already extensively studied and applied for amperometric, enzymatic, immuno and genosensing assays [45–47]. The uneven surface of the graphite–epoxy electrode allows the immobilization of DNA on its surface by simple physical adsorption. If needed, the electrode surface may be renewed after each experiment by polishing with abrasive paper [48]. This type of sensor has been already used for impedimetric detection of DNA hybridization [29]. Present work proposes the amplification of the impedimetric signal, as obtained in previous studies, by the use of strept-AuNPs; these are used after the hybridization step to bind to the biotinylated DNA hybrid. The biotinylated DNAnanoparticles conjugates formed, are responsible of impedance signal enhancement. The further silver deposition on streptAuNPs surface allowed the direct observation of the phenomena with scanning electron microscopy (SEM); the same process may be used to provide additional signal amplification. A competitive binding assay [49] using non-labelled DNA target was also performed with the aim of showing the viability of the procedure for specific gene determination in appropriate samples.

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• d(pT)20 (20 mer): 5 -TTTTTTTTTTTTTTTTTTTT-3 ; • d(pA)20 (20 mer): 5 -AAAAAAAAAAAAAAAAAAAA-3 ; • d(pA)20 -bio (20 mer): 5 -AAAAAAAAAAAAAAAAAAAA-3 (modified with biotin in 5 end); • d(pC)20 -bio (20 mer): 5 -CCCCCCCCCCCCCCCCCCCC3 (modified with biotin in 5 end). GECs were prepared using 50 ␮m particle size graphite powder (BDH Laboratory Supplies, Poole, UK) and Epotek H77 resin and hardener (both from Epoxy technology, Billerica, MA). 2.3. Construction of working electrodes GEC working electrodes were prepared by mixing graphite powder and epoxy resin in a 1:4 (w/w) ratio. The resulting paste was used to fill the inner cavity of a plastic body (6 mm i.d.) with an electrical internal contact [29]. The composite material was then cured at 80 ◦ C for 2 days. Before each use, the surface electrode was smoothed with abrasive paper (grades 400–1000) and finally with alumina paper (polishing strips 301044-001, Thermoelectron, Bremen, Germany). The reproducibility of construction and polishing of sensors based on GECs have been reported previously [50].

2. Experimental 2.4. DNA biosensing protocol 2.1. Apparatus An IM6e Impedance Measurement Unit (BAS-Zahner, Germany) was used for EIS measurements. A three-electrode cell was used, comprising a platinum auxiliary electrode (Crison 52-67 1, Barcelona, Spain), an Ag/AgCl reference electrode, i.e. an AgCl covered silver wire and the constructed graphite epoxy composite (GEC) as working electrodes. Temperature-controlled incubations were performed in an Eppendorf Thermomixer 5436. A scanning electron microscope (SEM) (Hitachi S-570, Tokyo, Japan) was used to visualize silver enhanced strept-AuNPs on electrode surface.

The immobilization of oligonucleotide probe onto the electrode surface was realized by simple physical adsorption. The hybridization was carried out incubating the electrode already modified with the probe in a solution containing the biotinylated complementary target. After the hybrid formation the sensor was incubated in a solution containing streptAuNPs, and DNA bound to nanoparticles through the formation of biotin–streptavidin complex. This step was followed by the silver enhancement treatment and the observation of the modified electrode surface with SEM. The scheme of experimental procedure, described in detail below, is represented in Fig. 1.

2.2. Chemicals and reagents Potassium ferricyanide K3 [Fe(CN)6 ], potassium ferrocyanide K4 [Fe(CN)6 ] and streptavidin-gold nanoparticles (Ref. S9059) were purchase from Sigma (St. Louis, MI). LI Silver Enhancement Kit was obtained from Nanoprobes (Yaphank, NY). Other reagents were commercially available and were all of analytical reagent grade. All solutions were made up using doubly distilled water. The following buffers were employed: 0.1 M PBS (0.1 M NaCl, 0.01 M sodium phosphate buffer, pH 7.0), TSC1 (0.75 M NaCl, 0.075 M trisodium citrate, pH 7.0), TSC2 (0.30 M NaCl, 0.030 M trisodium citrate, pH 7.0). Oligonucleotides used in this work were purchased from TIBMOLBIOL (Berlin, Germany). All DNA stock solutions were prepared with sterilized and doubly distilled water and kept frozen until used. Their bases sequences were:

2.4.1. DNA probe immobilization The dry adsorption procedure consisted of depositing 20 ␮l of probe oligonucleotide solution (at desired concentration in TSC1 buffer) onto the electrode surface and allowing its adsorption during 45 min, at the controlled temperature of 80 ◦ C. This was followed by a washing step with TSC2 buffer thus eliminating all adsorbed salt. 2.4.2. Incubation with DNA target GEC electrodes modified with DNA probe were incubated in an eppendorf tube containing 30 pmol of biotinylated complementary target oligonucleotide in TSC1 buffer solution (total volume 140 ␮l). The tube was then incubated at 42 ◦ C with a gentle stirring during 30 min. This step was followed by two gentle washing steps in TSC2 buffer for 5 min at 42 ◦ C. For ‘competitive assay’ experiments the concentration of biotinylated DNA target was fixed at 30 pmol, varying at the same time

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Fig. 1. Scheme of experimental procedure.

the concentration of non-labelled DNA target. Conditions such as type and concentrations of buffers and times involved in each different step (dry adsorption, hybridization and washing steps) were optimized previously [9]. 2.4.3. Addition of strept-AuNPs GEC electrodes modified with biotinylated hybrid were incubated in an eppendorf tube containing a solution of strept-AuNPs at desired concentration in PBS buffer. The tube was then incubated at 42 ◦ C with a gentle stirring during 20 min. This step was followed by two gentle washing steps in PBS buffer for 10 min at 42 ◦ C. Negative controls were performed for streptAuNPs addition step either using d(pC)20 -bio as biotinylated non-complementary target or d(pA)20 as non-biotinylated complementary target. 2.4.4. Silver enhancement of strept-AuNPs Twenty microlitres of a solution obtained by the combination of equal volumes of enhancer and initiator were deposited onto the electrode surface and left 7 min for the reaction. The electrodes were then thoroughly washed with deionized water to stop the reaction. The silver enhancing solution was always prepared immediately before use. For silver enhancement treatment, negative control consisted of using non-biotinylated complementary target. 2.5. Impedimetric detection Impedance measurements were recorded between 50 kHz and 0.05 Hz, at sinusoidal voltage perturbation of 10 mV amplitude and a sampling rate of 10 points per decade above 66 Hz, and five points per decade at the lower range. The experiments were carried out at applied potential of 0.17 V (vs. Ag/AgCl reference electrode) in a 0.1 M PBS buffer solution containing 0.01 M K3 [Fe(CN)6 ]/K4 [Fe(CN) 6 ] (1:1) mixture, used as a redox marker.

The theoretical curve used to fit the experimental data corresponds to an equivalent circuit formed by a capacitor and a resistor in parallel, both in series with another resistor. The chisquare goodness of fit was calculated for each fitting by the FRA software employed (Eco Chemie, The Netherlands). In all cases impedance data were registered in the following order after each electrode successive modification: (1) bare GEC electrode (blank); (2) probe immobilization; (3) biotinylated-target addition; (4) strept-AuNPs addition; (5) silver enhancement. 3. Results and discussion 3.1. DNA hybridization detection and signal amplification In Fig. 2, Nyquist plots obtained in a whole experiment of DNA biosensing are represented. The bare electrode was successively modified with: (1) DNA probe; (2) DNA target; (3) strept-AuNPs; (4) silver enhancement treatment. From the obtained signals, single semicircles, the equivalent circuit proposed to fit the experimental data is shown in Fig. 3. In the circuit, the parameter R1 represents the resistance of the solution; R2 (also called Rct ) corresponds to resistance to the charge transfer between the solution and the electrode surface; and CPE is associated to the capacitance of the double layer (due to the interface between the electrode and the electrolytic solution). The use of a constant phase element (CPE) instead of a capacitor is required to better fit the experimental data. In fact, observing the obtained spectra, we can notice depressed semicircle shapes. This behaviour is normally expected for electrodes made of composite material. For this reason, a CPE was used in the equivalent circuit to reflect the properties of non-ideal electrode surface [13,17]. The upward curvature observed in the spectra at low frequencies is due to the contribution of the diffusion. This factor was not taken into account since we focused

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Fig. 2. Nyquist diagrams for EIS measurements of: (filled circles) bare GEC electrode, (empty circles) probe modified electrode, (filled triangles) biotinylated-hybrid modified electrode, (empty triangles) biotinylated-hybrid modified electrode + strept-AuNPs, (filled squares) biotinylated-hybrid modified electrode + silver enhanced strept-AuNPs. All measurement were performed in 0.1 PBS buffer solution containing 10 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ]. The arrow in each spectrum denotes the frequency (AC) of 1.11 Hz.

on the charge transfer resistance (Rct ) between the solution and the electrode surface. A modification of the electrode surface strongly influences the latter, thus leading to a change in the Rct value (Rct corresponds in the spectra to the diameter of the semicircle). For this reason, it is possible to monitor each step of the biosensing just following the variation of this parameter. Besides, the change of time constant of the semicircles on modification was also monitored but it was not significant. The related difference of frequencies at the apex of the semicircle was within ± one experimental step of scanned frequency. For all performed fittings, the chi-square goodness-of-fit test was thoroughly checked to verify calculations. In all cases, calculated values for each circuit remained in the range of 0.0003–0.15 much lower than the tabulated value for 50 degrees of freedom (67.505 at 95% confidence level). As shown in Fig. 2, Rct increased after any further modification of the electrode surface. This can be attributable to the increased difficulty of the redox reaction of [Fe(CN)6 ]3−/4− to take place, due to the sensor surface alteration [15]. Two different factors may be taken into account to properly explain that: the electrostatic repulsion and the sterical hindrance. The former is more significant in the first and second step of the protocol: when DNA probe is immobilized onto the electrode surface, a first layer is formed, where negatively charged phosphate groups of DNA skeleton are responsible of the electrical repulsion towards the negatively charged redox marker, thus inhibiting the interfacial charge transfer process and resulting in Rct increment. The

Fig. 3. Equivalent circuit used for data fitting of EIS measurements.

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Fig. 4. Histograms representing comparison between experiments and two different negative controls: 1st bar: experiment with complementary biotinylated target + strept-AuNPs; 2nd bar: negative control no. 1 with a non-complementary biotinylated target + strept-AuNPs; 3rd bar: negative control no. 2 with a complementary non-biotinylated target + strept-AuNPs (Δs = Rct(sample) − Rct(blank) ; Δp = Rct(probe) − Rct(blank) ). Error bars correspond to triplicate experiments and are referred to the whole bar value (i.e. to delta ratio after nanoparticles addition).

addition of a second DNA strand to form DNA hybrid results in further increment of resistance value due to the improved quantity of negative charges and to the hindrance caused by the formation of a double layer. Hence, DNA hybridization can be proved by this further increment of charge transfer value [28]. In this work, a biotinylated oligonucleotide was used as complementary target, with the aim of amplifying the EIS hybridization signal by the addition of strept-AuNPs. As shown in Fig. 2, after the addition of strept-AuNPs we can observe a further increment of Rct value because of the increased space resistance due to Au–streptavidin conjugates. At working pH 7, streptavidin is slightly negatively charged [51] (pI is around pH 5) and this fact also contributes to enhance the resistance in consequence of electrostatic repulsion with redox marker. In order to observe the presence and distribution of streptAuNPs, a Silver enhancement treatment [44,52,53] was applied to electrodes already modified with DNA–nanoparticle conjugates. EIS measurement was performed also after this treatment and, as shown in Fig. 4, a significant increment of Rct value was observed, attributable to silver deposition on gold. The results are expressed as the relative Rct variation between the values obtained in the different experiments (i.e. DNA adsorption, hybridization, strept-AuNPs addition and silver enhancement treatment) and Rct value due to the bare electrode. This relative variation is represented as a ratio of delta increments (see delta ratio Δs /Δp , footnotes of Table 1). The elaboration required for the obtained data have been already used and extensively explained in previous work [29]. Δs /Δp value should be >1 for the hybridization experiments and ≈1 for negative controls with no complementary targets (that means Δs = Δp , i.e. no variation of Rct value after hybridization). In this way it was possible to reduce the contribution of GCEs construction and polishing to the relative standard deviation of the signal. In fact, from impedimetric studies on reproducibility, R.S.D.% due to the polishing procedure resulted to be less than 3.5% (experiment performed with the same electrode), whilst

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Table 1 Relationship between strept-AuNPs concentration and EIS signal obtained in experiments and negative controls Strept-AuNPs concentration

Experiment (Δg /Δp )a

Negative control (Δg /Δp )a

Relative percentage variationa

Stock solution 1:10 1:100 1:1000

4.02 (±0.10) 3.24 (±0.08) 2.76 (±0.05) 1.60 (±0.08)

2.40 (±0.12) 1.95 (±0.10) 1.42 (±0.09) 1.40(±0.09)

67% 66% 94% 14%

aΔ g

= Rct(hybrid+gold) − Rct(blank) . Relative percentage variation = [delta Δp = Rct(probe) − Rct(blank) . ratio(experiment) − delta ratio(negative control) ] 100/delta ratio (negative control) .

R.S.D.% coming from electrode construction was less than 6.7% (experiments performed with different electrodes). In the histogram, the first bar corresponds to Rct variation of a hybridization experiment with a complementary target and the further addition of Au–streptavidin conjugate; the second bar corresponds to the first negative control where biotinylated noncomplementary target was used in the hybridization step (and then strept-AuNPs were added as before); the third bar represents the results obtained with second negative control, where a nonbiotinylated complementary target was used in the hybridization step (and then strept-AuNPs were added as before). As it can be observed in the graph, the highest delta ratio was obtained when a complementary biotinylated DNA target is used. The first negative control was performed to demonstrate that if a non-complementary target is used, the signal variation during the hybridization step can be considered not significant (double stranded DNA is not formed). However, the increment obtained for strept-AuNPs addition was noticeable (it represents in fact the 29% of the signal, RSD% = 8.5), while it may be explained by certain non-complementary target not specifically adsorbed onto the electrode surface. In fact, biotin molecules of this noncomplementary oligomer strongly bound to strept-AuNPs thus incrementing Rct value. Nevertheless, this negative control was sufficiently different form the reference, where the resulting amplification was 70% larger. A second negative control was carried out to show that adsorption alone of strept-AuNPs on the electrode surface was negligible. If a complementary non-biotinylated target was used, strept-AuNPs did not remain on the electrode surface. According to obtained results, change of delta ratio after addition of strept-AuNPs was not significant. 3.2. Optimization of DNA probe concentration EIS measurements with different amounts of probe oligonucleotide were carried out in order to optimize the DNA probe concentration to be used in the protocol. This concentration should ensure a full coverage of the electrode surface, avoiding any possible non-specific adsorption of both DNA target and strept-AuNPs. The obtained results are shown in Fig. 5. In there, the variation (Δp ) of charge transfer resistance between blank and probemodified electrode is plotted versus DNA probe concentration. The increment of DNA probe concentration accompanied the

Fig. 5. Calibration curve obtained with GEC electrodes modified with different amounts of DNA probe. All DNA sequences were diluted in 140 ␮l of buffer solution. (Δp = Rct(probe) − Rct(blank) ). Error bars correspond to triplicate experiments.

enhancement of Rct value until a plateau is reached. At this point, the electrode surface can be considered completely covered by the immobilized oligonucleotide and any additional increment of DNA did not result in a further increment of Rct value. Therefore, DNA probe of 30 pmol was chosen for the successive experiments. 3.3. Optimization of strept-AuNPs concentration A comparison among signals obtained from the experiments and negative controls was made in order to find optimum concentration conditions. Values employed were: stock solution without any dilution; 1:10, 1:100, and 1:1000 (v/v) dilutions of the stock solution in PBS buffer. Table 1 summarizes the results obtained in these experiments. As it can be observed, when the concentration of streptAuNPs was decreased, the variation of charge transfer resistance, expressed as delta ratio, resulted lower. The highest signal corresponded to the more concentrated solution (stock solution). Additionally, the signal obtained was compared with the negative control assays (where non-biotinylated DNA target was used during hybridization step). In the latter, strept-AuNPs were not supposed to bind to non-biotinylated DNA oligonucleotides, but observing the results some non-specific adsorption of strept-AuNPs onto the electrode surface is present in all cases. This non-specific adsorption, depending on the uneven surface of GEC electrode [48], resulted lower at lower concentration of initial solution employed. The optimal strept-AuNPs concentration to be used in the experiments was therefore the one ensuring the signal amplification with the lowest non-specific adsorption. This was a 1:100 dilution from the initial stock solution which corresponded to the highest net percentage variation of signal. 3.4. Scanning electron microscope characterization GEC electrode surfaces were inspected by SEM after Silver Enhancement treatment (an acceleration voltage of 15 kV and a resolution of 5 ␮m were used to take the different images). LI Silver reagent was employed in order to obtain an adequate

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Fig. 6. SEM images of (a) experiment using probe + biotinylated complementary target + silver enhanced strept-AuNPs; (b) negative control using probe + nonbiotinylated complementary target + silver enhanced strept-AuNPs. All images were taken at an acceleration voltage of 15 kV and a resolution of 5.0 ␮m.

enlargement of strept-AuNPs present on the sensor surface to allow their observation with SEM. The images obtained are shown in Fig. 6. On part (a), a biotinylated complementary target was used during the hybridization step. In there, the strept-AuNPs distribution over the electrode surface is quite homogeneous. Also, comparing this image with the negative control that did not use biotinylated complementary DNA target – part (b) –, it is clear that streptAuNPs bound mostly to biotin-modified DNA; additionally, it may be deducted that non-specific adsorption was much reduced and nearly negligible. 3.5. Entity of signal amplification To quantify the amplification of the signal obtained with this protocol, a calibration curve was performed, using a fixed concentration of DNA probe, versus biotinylated DNA target. The results obtained with these experiments are shown in Fig. 7 (filled circles). The increment of target concentration led to the increment of Rct , and resulting in a dynamic response

Fig. 7. Calibration curve obtained with GEC electrodes modified with (a) different amounts of DNA biotinylated-hybrid + strept-AuNPs (filled circles); (b) different amounts of DNA non-biotinylated (filled squares). All DNA sequences were diluted in 140 ␮l of buffer solution (Δs = Rct(sample) − Rct(blank); Δp = Rct(probe) − Rct(blank) ). Error bars correspond to triplicate experiments.

range (non-linear) between 2 and 30 pmol. After this point, a plateau was reached and a further increment of target amount did not cause any improvement. If we compare these results with those obtained without strept-AuNPs amplification step (filled squares), we can observe that the same variation of Rct value (expressed as delta ratio) was obtained when a higher amount of DNA target was employed. In fact, as shown in the figure, a delta ratio value of about 1.3 was measured with 30 pmol of DNA target in experiments without amplification, while almost the same delta ratio (ca. 1.4) resulted with a target concentration of only 5 pmol in experiments with strept-AuNPs. This means that we can obtain the same signal with a DNA concentrations 4–5 times lower. Results with an amount of DNA target smaller than 2 pmol are not reported because they presented a degraded reproducibility. 3.6. Competitive binding assay The quantification of non-labelled DNA target was realized using a competitive binding assay protocol. The aim of this experiment was to quantify unknown amount of unlabelled DNA in presence of a fixed amount of competing biotinylated ssDNA competitor [49]. The results obtained with these experiments are represented in Fig. 8. As shown in the graph, signal variation at low concentration of unlabelled DNA target (left part of the figure) is comparable to the one obtained in the calibration curve in which only biotinylated DNA target was present (see Fig. 7). Increasing the concentration of unlabelled ssDNA in the hybridization step, the competition between the two targets led to a decrement of the signal. This was due to the lower amount of biotinylated DNA hybridizing with DNA probe. A further increment of unlabelled DNA target concentration caused an additional decrease and stabilization of the signal. This experiment was performed in order to apply the method to future detection of DNA from real samples, which preferably

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Project CSD2006-00012) and by the Department of Innovation, Universities and Enterprise (DIUE) from the Generalitat de Catalunya. References

Fig. 8. Competitive binding assay performed when fixing the concentration of biotinylated DNA target at 30 pmol value and increasing the concentration of unlabelled DNA target.

should be unlabelled. In fact, as deducted from the graph, the protocol permitted detection of sample DNA around 16 pmol (calculated as EC50 in the graph). 4. Conclusions In this work, the signal amplification of impedimetric detection of DNA hybridization is proposed using gold-streptavidin nanoparticles (strept-AuNPs). The coating with streptavidin favoured the rapid formation of conjugates with biotinylated DNA hybrid, thus avoiding the need of synthesizing DNA modified with strept-AuNPs, a more complex procedure. Besides the good reproducibility of obtained results (R.S.D.% less than 8.5%), it was possible to increase the sensitivity of the method, obtaining comparable signals with a DNA amount ca. five times lower. For a comparable amount of DNA, the signal resulted 90% amplified, compared with results recorded without the use of strept-AuNPs. Moreover, the limit of detection obtained with this new protocol (11.8 pmol, S/N = 3) is also improved when compared to our previous work without amplification (26.5 pmol, S/N = 3). The demonstration of a competitive binding assay opens novel avenues for future applications in the analysis of unlabelled DNA sequences coming from real samples. A Silver Enhancement treatment was also performed to visualize the gold nanoparticles on the electrode surface with SEM, causing strept-AuNPs to grow from 10 nm to ca. 500 nm. This treatment allowed a clear visualization of the particle distribution on the sensor surface and an estimation of the non-specific adsorption. This may be reduced if the appropriate concentrations of reagents were employed. Acknowledgments Financial support for this work has been provided by the Ministry of Education and Science (MEC, Madrid, Spain) trough project NANOBIOMED (Consolider-Ingenio 2010,

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