Amplified electrochemical genotyping of single-nucleotide polymorphisms using a graphene–gold nanoparticles modified glassy carbon platform

Abstract

The main challenges in the construction of DNA biosensors for the genotyping of all the possible single-nucleotide polymorphisms (SNPs) are their sensitivity, speed and cost. The application of nanoparticle-modified monobases for the electrochemical genotyping of SNPs has been investigated in our previous study. In the present manuscript, that strategy was modified by applying a graphene–gold nanoparticle (GR–AuNPs) nanocomposite to achieve further amplification and higher sensitivity of SNPs genotyping. The present strategy shows a good potential for sensitively discriminating, quantifying and genotyping different SNPs. Taking the advantages of triple-amplification effects of AuNPs, GR and modified metal (Au and Ag) nanoparticles, this DNA biosensor exhibits highly sensitive responses for the genotyping of different SNPs and the detection of thermodynamically stable SNP (G–T) and A–C mismatch targets in the range of 10–1700 pM and 20–1200 pM with the detection limits of 2 and 10 pM (3σ) for G–T and A–C mismatch targets, respectively. The results demonstrate that when the surface coverage of DNA per unit area is just slightly increased, there is a dramatic increase in the active surface area, and the absolute loading amount of DNA on the surface would also be increased.

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1. Introduction

There is an urgent need to develop sensitive, selective, and rapid strategies for SNP detection in mammalian genetics as well as in clinical practices. SNPs are single-base mutations that can occur in coding regions and may lead to major health problems, genetic diseases and altered response to drug treatments.¹ Applications of different SNP genotyping technologies, especially in biomedical areas and clinical diagnostics, have been thoroughly discussed in numerous excellent reviews.^2–4 Moreover, high-throughput genotyping is traditionally performed using next-generation sequencing^5–7 as a sensitive diagnostic method. However, this technique is relatively costly, time consuming and labor intensive. Among these technologies, electrochemical genosensors offer a promising alternative for carrying out the applications of SNPs analysis due to their low cost, miniaturization, high sensitivity, and compatibility with micro-fabrication technology.^8–10 The development of methods, which require more sensitive, inexpensive and simpler protocols, is very important in the genotyping of different SNPs and though electrochemical genosensors that have been used for the coding of SNPs possess the abovementioned characteristics, to date, they have been reported only in few manuscripts. Moreover, to meet the increasing demand for the ultrasensitive biosensing detection of low-abundance SNPs, various signal amplification strategies, including the application of enzymes,¹¹ apoferritin,^12,13 liposomes,¹⁴ nanoparticles,^15,16 and quantum dots,^17,18 have been employed. Willner's group used a monobase-modified alkaline phosphatase enzyme and also liposomes for the detection of single base mutations.^11,14 Liu and Lin¹² described the novel quantification of SNPs using the monobase-modified cadmium phosphate loaded apoferritin. However, enzyme or protein-based detections are limited due to the denaturation and leakage of nanoparticles, high-cost and time-consuming purification process. Monobase-modified gold nanoparticles, as a redox probe, have been used by Kerman's group for the genotyping of SNPs.¹⁵ However, the electrochemical oxidation of AuNPs occurs at a high positive potential, close to the potential at which guanine and adenine undergo oxidation, which can limit the precision and selectivity of the assay.¹⁹ In another reported strategy for the coding of SNPs, Zhang and his co-workers¹⁷ proposed a method for the detection of point mutation using different single-base-coded quantum dot (QD) nanoparticles. In these methods, despite the advanced detection strategies, relatively little progress in the electrochemical simultaneous genotyping of multiple significant genetic variations has been observed. Subsequently, different nanocrystal quantum dots,¹⁸ apoferritin loaded with different metal nanoparticles,¹³ and monobase-modified silver and gold nanoparticles¹⁶ have been investigated. However, the QD-based detections need complicated syntheses and necessitate severe detection conditions such as the dissolution of nanocrystals and accumulation of high potentials, which are not suitable for the routine analyses. Other critical points to further enhance the sensitivity of the biosensors are the reduction of background current to improve signal-to-background ratio on one side and increase in the loading amount of self-assembled sensing monolayer on the other side. Graphene oxide (GO) and GR along with silver and gold nanoparticles (Ag/AuNPs) have received tremendous attention owing to their unique properties such as excellent conductivity, high surface area, catalytic properties, electron transfer enhancement, non-toxic nature and good biocompatibility. Therefore, they have been applied in the design and fabrication of different electrochemical immunosensors and DNA sensors to amplify the analytical signals.^20–26 For example, Zhang's group reported an electrochemical DNA hybridization biosensor using gold nanorods (AuNRs) and graphene.^27,28 GR–AuNPs have also been used for the detection of oxygen reduction as well as glucose,^29,30 dopamine,³¹ phoxim,³² carbamazepine,³³ sumatriptan,³⁴ thrombin,³⁵ bisphenol A,³⁶ ractopamine³⁷ and nitrite.³⁸

Electrodeposition technique is an effective and simple method for synthesizing metal or alloy NPs.³⁹ Moreover, recently, the electrochemical reduction of GO to GR has drawn great attention as an effective tool because it is fast and involves green and non-toxic solvents.⁴⁰ Therefore, the electrodeposition of metal NPs has been used for higher loading of different NPs on a GR-modified electrode.^41,42 Our group recently reported an electrochemical simultaneous SNPs genotyping using monobase-conjugated modified nanoparticles on a single platform.¹⁶ In addition, in another study, dual amplification SNPs using a nanoporous gold electrode and GO was recently reported by our group.⁴³ In continuation of our previous study, to amplify the signal, a sensing platform for an ultrasensitive, selective, and efficient detection of SNPs based on graphene–AuNPs modified glassy carbon electrode (GR–AuNPs/GCE) has been developed. Herein, for genotyping of individual SNPs, the same strategy that was developed by our research group has been used.¹⁶ Briefly, first, GR was immobilized on a pretreated GCE. Then, AuNPs were electrochemically deposited on the GR modified GCE (GR/GCE) for the effective and directional immobilization of the sensing interface (probe oligonucleotide) via gold–sulfur chemistry. SNPs were detected by hybridization of mismatch targets using complementary monobase modified Ag and AuNPs in the presence of DNA polymerase I (Klenow fragment) via the following electrooxidation signal of AgNPs and 3,4-diaminobenzoic acid (DABA) as signal tracers. Polymerase enzyme induces coupling of the monobase modified nanoparticles to the mutant site of duplex DNA. Triple signal amplification for electrochemical SNPs genotyping was developed by the following processes: (1) GR accelerates the electron transfer rate,^19,44 (2) AuNPs increase the surface area for loading a high-content of sensing interface because the intrinsic property of high surface-to-volume ratio of AuNPs and GR–AuNPs hybrid as a sensing platform has a synergetic effect for improving the performance and (3) large amounts of AgNPs and DABA as signal tracers prompt the sensitivity. To demonstrate the substantial role of GR in signal amplification, the signals of the biosensor based on AuNPs/GCE and GR–AuNPs/GCE platforms were compared. They exhibited considerably higher electrochemical performance, which can be attributed to the high surface area and the excellent electrical properties of GR.

2. Experimental

2.1. Materials and apparatus

Synthetic oligonucleotides were purchased from Eurofins MWG/Oberon Co. with the following sequences (5′ to 3′):

Probe	SH–(CH2)6–CTG CGT TTT
Complementary	TGC CGA AAA AAA ACG CAG
A–C mismatch	TA̲C CGA AAA AAA ACG CAG
G–T mismatch	TGC T̲GA AAA AAA ACG CAG
Capture	TTT TCG GCA

Tris(2-carboxyethyl)phosphine (TCEP), Tris–HCl, sodium hydroxide (NaOH), sodium chloride (NaCl), potassium chloride (KCl), potassium nitrate (KNO₃), magnesium chloride (MgCl₂), potassium dihydrogen phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), hydrochloric acid (HCl), sulfuric acid (H₂SO₄), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), potassium ferrocyanide (K₄[Fe(CN)₆]), potassium ferricyanide (K₃[Fe(CN)₆]), sodium borohydride (NaBH₄), 3,4-diaminobenzoic acid (DABA), ruthenium hexamine trichloride (RuHex), cysteine (Cys), cysteamine hydrochloride (Cyt-HCl), potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂) and silver nitrate (AgNO₃) were purchased from commercial companies (Sigma Aldrich or Merck). The stock solutions of the oligonucleotides were prepared with 1× phosphate buffer solution (PBS 1×) of pH 7.4 (0.01 M Na₂HPO₄, 2 mM KH₂PO₄, 0.15 M NaCl and 0.15 M KCl). Adenosine 5′-triphosphate (ATP), cytidine 5′-triphosphate (CTP), guanosine 5′-triphosphate (GTP), thymidine 5′-triphosphate (TTP) and DNA polymerase I (Klenow fragment), were purchased from Vivantis Co. (Malaysia). Monobase solutions were prepared using a 20 mM Tris–HCl buffer solution containing (TBS) 20 mM NaCl (pH 7). A conventional electrochemical cell consisting of the modified glassy carbon electrode (GCE) in connection with a Ag/AgCl (3 M KCl) electrode and a Pt wire, as the working, reference and counter electrode, respectively, was used. The electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were carried out using an Autolab PGSTAT30 in the solution containing K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1 : 1, 0.5 mM) as the redox couple. EIS measurements were performed by applying an AC potential with a signal amplitude of 5 mV and a frequency range of over 10 kHz to 0.1 Hz at the open circuit potential (OCP). Differential pulse voltammetry (DPV) was used for electrochemically genotyping SNPs and for the quantification of thermodynamic G–T and A–C mismatches over a range of −0.2 to 0.6 V at the scan rate of 20 mV s⁻¹ and pulse amplitude of 25 mV. Field emission scanning electron microscopy (FE-SEM) was accomplished on a Mira 3-XMU at an accelerating voltage of 20 kV. X-ray diffraction (XRD) patterns of the samples were recorded on a Bruker D8/Advance X-ray diffractometer with Cu-K_α radiation at 40 kV and 40 mA. Raman spectra were collected on a SENTERRA Raman spectrometer using a 745 nm laser excitation. X-ray photoelectron spectroscopy (XPS) analysis was carried out using a VG Microtech Twin anode XR3E2 X-ray source and a concentric hemispherical analyzer operated at a base pressure of 5 × 10⁻¹⁰ mbar using Al K_α (hν = 1486.6 eV).

2.2. Procedure for electrochemical genotyping of SNP

2.2.1. Preparation of GR–AuNPs/GCE or AuNPs/GCE. Prior to the preparation of the modified electrode, GCE was mechanically polished with 1, 0.3, and 0.05 μm alumina powder sequentially to a mirror finish. For the preparation of GR–AuNPs/GCE, primarily 10 μl of 0.5 mg mL⁻¹ exfoliated GO was casted on a GCE surface, and electrochemical reduction of GO was performed to prepare GR/GCE by scanning the potential between 0 and −1.5 V for 25 cycles in PBS at pH = 4.0.⁴⁰ Then, the GR/GCE was immersed in a solution containing 0.01 g L⁻¹ HAuCl₄ and 0.1 M KNO₃ as a supporting electrolyte for the electrodeposition of AuNPs at a constant potential of −0.2 V for 90 s under magnetic stirring. For comparison with AuNPs/GCE, GCE surface was similarly treated in the absence of GR.

2.2.2. Electrochemical genotyping. After providing the platforms, biorecognition layers were prepared using the same strategy that was previously developed by our group.^16,43 The surface coverage of DNA on the surface was obtained using chronocoulometry. The characterization of surface platform for the immobilization of different DNA's was performed by EIS and voltammetric techniques.

3. Results and discussion

Graphene oxide (GO) was synthesized from graphite powder by a modified Hummers method,⁴⁵ which was reported in our previous manuscript.⁴³ Cysteine and cysteamine-modified silver and gold nanoparticles were also prepared using our previous study.¹⁶ Monobase-coded nanoparticles (M-NPs) probes, including thymidine-coded AgNPs (T-AgNPs), guanosine-coded AgNPs (G-AgNPs), cytidine-coded DABA-modified AuNP (C-Au-DABA), adenosine-coded DABA-modified AuNP (A-Au-DABA) and guanosine-coded DABA-modified AuNP (G-Au-DABA) were prepared via phosphoramidate bond of 5′-phosphate group of monobases with the free amino groups of the immobilized cysteine and cysteamine on the surface of AgNPs and AuNPs.¹⁶

3.1. Characterization of modified GCE

Fig. 1A shows the XRD patterns of the pristine graphite, GO, GR and GR–AuNPs. The diffraction peak of exfoliated GO with an inter-distance (d-spacing) of 7.75 Å appears at 11.4° (002). In comparison to the pristine graphite, this value is larger than the d-spacing (3.35 Å) of pristine graphite (2θ = 26.6°) due to the presence of oxygen containing functional groups.⁴⁰ After the electrochemical reduction of GO, the diffraction peak of GR appears at 26.6°, which is similar to previously reported results.⁴⁰ The XRD pattern shows obvious peaks located at the 2θ values of 39.6°, 46.3°, 67.4° and 81.4°, corresponding to the (111), (200), (220), and (311) planes, respectively. This confirms the presence of AuNPs on GR/GCE, and thus the formation of GR–AuNPs hybrid with the extensive conjugated sp² carbon networks, which is represented by a peak located at 26.2°, which corresponds to the interlayer spacing of 3.4 Å.⁴⁶ As shown in Fig. 1B, the Raman spectrum of GO contained both D and G bands at 1360 and 1585 cm⁻¹, which are representatives of sp³ and sp² carbon hybridization, respectively. After the electrochemical reduction of the exfoliated GO, the intensity of D band and ratio of D/G increased due to a decrease in the average size of sp² carbon domains of GO.⁴⁰ Fig. 1C shows the SEM image of GR–AuNPs, which shows that AuNPs are uniformly covered on the GR with an average size of 20 nm.

Fig. 1 XRD pattern of graphite, GO, GR and GR–AuNPs (A), Raman spectra for GO and GR (B), SEM image of GR–AuNPs (C).

The chemical and the content elemental composition were characterized using XPS.⁴⁷ The XPS survey spectrum of the GR–AuNPs nanocomposite showed distinct Au 4f, C 1s and O 1s peaks (Fig. 2A) without any other impurities.^48,49 The high-resolution deconvoluted C 1s spectra of GO (Fig. 2B) and GR (Fig. 2C) samples reveal the presence of C–C (284.3 eV), C–O (286.3 eV), C=O (287.7 eV), and O–C=O (289.0 eV).^45,50 Although the C 1s XPS spectra of GR and GO also exhibit the same species, the peak intensities of oxide species for the GR are considerably weaker than those observed in the spectrum of GO. These results confirm that O/C ratio in the exfoliated GO (32.5%) compared to GR (7.8%) remarkably decreases after the electrochemical reduction, i.e., ∼76% of most epoxide and hydroxyl functional groups have been successfully removed during the electrochemical reduction. The XPS analysis was performed on the GR–AuNPs nanocomposite platform to obtain the content and information about the oxidation state of AuNPs.^51,52 Fig. 2D shows a pair of doublet peaks at 85.3 and 89.1 eV, which correspond to metallic Au atoms (Au⁰). Furthermore, the absence of peaks for Au⁺ species at 86.2 and 89.9 eV demonstrates that AuNPs exists is in metallic form on the GCE surface. The AuNPs content in the GR–AuNPs nanocomposite platform was found to be ∼16.3%, as estimated from the XPS data.

Fig. 2 XPS spectra of GR–AuNPs nanocomposite (A); high-resolution C 1s XPS spectra of GO (B) and GR (C); and Au 4f region (D).

3.2. Electrochemical performance of different modified electrodes

To confirm that the modified GCE has been constructed successfully, each modification step was investigated using CV and EIS experiments. The CVs were recorded over a range of −0.2 V to +0.6 V in a solution containing [Fe(CN)₆]^3−/4−on bare GCE, GR/GCE, AuNPs/GCE and GR–AuNPs/GCE, as shown in Fig. 3A. For the bare GCE, a pair of well-defined peaks with ΔE_p = 79 mV are observed. However, the peak currents of redox probes increased in each modification step of GCE with GR, AuNPs and GR–AuNPs. In addition, the largest peak currents and reversible behavior (ΔE_p < 65 mV) were related to the GR–AuNPs/GCE, indicating that the electron transfer rate increased on GR–AuNPs/GCE. Moreover, the corresponding electrochemical impedance spectra of different modified electrodes were studied. Fig. 3B shows the impedance spectra after each GCE modification step. The R_ct of the redox probe on the bare GCE (spectrum a) is 720 Ω. After the modification of GCE with AuNPs, GR and GR–AuNPs composite (spectra b, c and d, respectively), the R_ct decreases to 630, 360 and 250 Ω, and therefore faster electron transfer rates compared to bare GCE are observed, which are in accordance with the result of CVs. In particular, when AuNPs are electrochemically deposited on the surface of the GR/GCE, the R_ct of GR–AuNPs/GCE decreased even more than GR/GCE and AuNPs/GCE, demonstrating the synergistic effect of AuNPs and GR. These results suggest that GR–AuNPs/GCE can provide a unique platform for the genotyping of mismatches.

Fig. 3 (A) CVs of different GCE modification steps in the presence of 0.5 mM [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ (equimolar) at a scan rate of 100 mV s⁻¹. (B) EIS of 0.5 mM [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ (equimolar) for different modified electrodes: (a) bare GCE, (b) AuNPs/GCE, (c) GR/GCE and (d) GR–AuNPs/GCE.

3.3. Electrochemical genotyping of different mismatches

The general concept for the amplified detection of a single-base mismatch in the DNA is illustrated in Scheme 1. After the electrodeposition of GR and AuNPs on the GCE, the hybridization of the immobilized probe on the GR–AuNPs/GCE with the different concentrations of mutant (A–C and G–T) or complementary targets generate the respective double-stranded form on the electrode surface. Finally, the non-hybridized section of the target is hybridized with the capture strand. Then, the resultant structure was treated by M-NPs, leading to the hybridization of M-NPs with the mutant sites, and no interaction was observed in the presence of complementary target. The assembled probe on the modified electrode surface was examined by a chronocoulometric method,⁵³ showing a surface coverage of 4 (±0.5) × 10¹² molecules per cm². The surface coverage of the electrode given as DNA probe per unit area does not change significantly by increasing the surface area by applying GR–AuNPs, while the loading amount of the probe on the surface is increased.

Scheme 1 Schematic illustration of amplified electrochemical genotyping of different SNPs.

Because the density of AuNPs on the GCE strongly influences the detection sensitivity of a genosensor, it is of key importance. To investigate the effect of the density of AuNPs on electrode responses, the electrodeposition of AuNPs on the GR/GCE was carried out at different deposition times. By increasing the density of AuNPs, the analytical signals of the genosensor in the presence of mismatched targets increase and reach to the maximum amount at the deposition time of 90 s (Fig. S1, curve c, ESI†). However, further increase in the deposition times does not increase the analytical signals, but lead to decrease in the electrooxidation currents of AgNPs and DABA (Fig. S1, curve d, ESI†). This phenomenon can be attributed to the increases in the size of the nanoparticles as well as their aggregation, and therefore it leads to a decrease in the loading amount of DNA and subsequently lower loading amounts of molecular reporters, i.e. AgNPs and DABA molecules, on the surface.

Fig. 4 shows a set of DP voltammograms for the detection of A–C and G–T mismatches. As expected, by the binding events illustrated in Scheme 1, A–C and G–T mismatches are hybridized with their complementary bases in modified M-NPs, i.e., with T-AgNPs/G-AgNPs and C-Au-DABA/A-Au-DABA, respectively. After the A–C modified electrode was subjected to T-AgNPs/G-AgNPs, a well-defined oxidation peak of AgNPs was observed because of the hybridization of adenosine and cytosine bases of A–C mismatch with complementary bases of T and G and accumulation of AgNPs on the electrode surface. Moreover, the electrooxidation of DABA was followed for the genotyping of G–T mismatches after coding by C-Au-DABA/A-Au-DABA. Fig. 4 compares the DPV responses of the electrooxidation of signal tracers on the bulk gold electrode,¹⁶ AuNPs/GCE and GR–AuNPs/GCE for the genotyping of A–C and G–T mismatches. As shown in Fig. 4A, for the genotyping of A–C mismatches on the AuNPs/GCE, the anodic peak current (I_pa) of AgNPs is found to be 1.6 μA, while the GR–AuNPs/GCE gives an I_pa of 3.4, which is around 2-fold higher than that of the AuNPs/GCE. Moreover, the comparison between the anodic peak currents of this protocol and those previously reported by us on the bulk gold electrode¹⁶ demonstrates that the signal has been amplified 3.2 times at the same concentration (800 pM).

Fig. 4 (A) Electrooxidation of AgNPs for the genotyping of A–C mismatch target; (B) electrooxidation of DABA for the genotyping of G–T mismatch target (C) simultaneous genotyping of A–C mismatches after interaction with T–AgNPs/G-Au-DABA. (D) Simultaneous genotyping of complementary target in the presence of T-AgNPs/G-Au-DABA (on bulk gold, AuNPs/GCE and GR–AuNPs/GCE surfaces).

In addition, the electrooxidation of DABA was monitored after the treatment of G–T mismatch-modified electrode with C-Au-DABA/A-Au-DABA (Fig. 4B). The differential voltammogram of DABA accumulated on the bulk gold electrode after modification with 800 pM G–T mismatches shows a small response, whereas the hybridization of AuNPs/GCE modified electrode with the same concentration of G–T mismatch targets shows a relatively higher peak current. To demonstrate the efficiency of genotyping to improve the detection sensitivity, the GR–AuNPs/GCE was also used as a modified platform, which displayed a considerably higher peak current. The high performance response of GR–AuNPs nanocomposite platform can be attributed the combination of the following factors: (1) higher loading amount of DNA probe using AuNPs; (2) the electrical network of AuNPs because of their direct incorporation with the GR;²⁹ (3) a considerably easier accessibility of M-NPs to mutant sites; and (4) the high-efficiency electron acceleration of the GR.⁵⁴ GR sheets serve as the electron acceptor and transporter.^55,56 Therefore, the well-distributed AuNPs on the surface of graphene would induce more active sites for the immobilization of molecular reporters. Moreover, this leads to an efficient electrical network through their direct incorporation with GR. Moreover, by the electrodeposition of GR on the GCE, the electrode surface reveals a thin layer with a typical crumpled and wrinkled structure.⁵⁷ Therefore, AuNPs are electrochemically deposited on an uneven surface rather than on the smooth bare GCE. This suggests that the immobilization of DNA probes and subsequently the hybridization of the targets and capture are performed in different layers, and the accessibility of M-NPs to the mutant sites on the mismatch targets is considerably easier. Consequently, larger amounts of M-NPs are accumulated on the mismatch-modified electrode, and this leads to higher response currents. Moreover, Xu et al.⁵⁴ recently reported that when the GO is electrochemically reduced to GR, it has a much higher electron transfer rate in comparison with chemically reduced GO. The fast electron transfer should be attributed to the edge effect of GR due to the crumpled and wrinkled structure.

The oxidation potential of AgNPs and DABA are completely separated (0.2 and 0.44 V, respectively), and it would be expected that the biosensor can be used simultaneously for different SNPs genotyping using AgNPs and DABA. For this purpose, e.g. A–C mismatches, the modified electrode was treated with T-AgNPs/G-Au-DABA (Fig. 4C). The simultaneous treatment of the electrode with T-AgNPs and G-Au-DABA shows well-resolved oxidation signals of AgNPs and DABA. Fig. 4D shows the modified platforms in the presence of completely complementary DNA when exposed to T-AgNPs/G-Au-DABA. No obvious signals can be observed on the different modified electrodes, especially on the GR–AuNPs/GCE platform; this demonstrates that the effect of the non-specific adsorption of M-NPs could be ignored. Distinct differences are observed in DPV responses between the complementary DNA and the A–C point mutation after adding T-AgNPs/G-Au-DABA to DNA duplex. These results imply that the hybridization has taken place through the specific base-pairing. Moreover, the intensities of currents in A–C and G–T mismatch targets when treated with T-AgNPs/G-AgNPs (Fig. 4A) and C–Au-DABA/A-Au-DABA (Fig. 4B), respectively, are almost twice the signal of A–C mismatches when the simultaneous detection is carried out (Fig. 4C) (treated with T-AgNPs/G-Au-DABA). It is worth noting that the background signals of the biosensor are not increased significantly by applying GR–AuNPs/GCE as the biosensor platform, resulting in the improvement of signal-to-noise in comparison to the bulk gold electrode.

Fig. 5 illustrates the comparison between the electrooxidation of AgNPs and DABA at the same concentration of A–C and G–T mismatches (800 pM), respectively, on the GR–AuNPs/GCE platform. As shown in Fig. 5, the proposed electrochemical biosensor using DABA as a signal tracer exhibits a larger current shift (∼ΔI = 3 μA) in comparison to AgNPs. Cysteamine-modified AuNPs were used as a carrier for DABA tracers. G–T mismatch targets form more thermodynamically stable double ^66,67strand form with hybridization efficiency considerably higher than that of A–C mismatch targets, since numerous DABA molecules could be loaded on the surface of AuNPs, which leads to a better analytical signal performance. Although the oxidation signal of AuNPs can be directly used as an analytical signal, their electrochemical oxidation occurs at a relatively high potential. In contrast, AgNPs oxidized at a more moderate potential, which resulted in an obviated interference species, and owing to a relatively sharp peak, sensitivity and selectivity are improved.

Fig. 5 Comparison between the electrooxidation of AgNPs (a) and DABA (b) on the GR–AuNPs/GCE surface.

Fig. 6 displays the electrochemical responses of different concentrations of thermodynamically stable G–T and A–C mismatch targets. The resulting calibration curves are linear (inset) in the range of 10–1700 pM and 20 to 1200 pM with the detection limits of 2 and 10 pM (3σ) for G–T and A–C mismatch targets, respectively. The signals are highly repeatable with a relative standard deviation of less than 5%. Therefore, the amplification of signals using the GR–AuNPs/GCE as a platform enables highly sensitive simultaneous electrochemical detection of any possible SNPs. The linearity of the calibration curve was verified using the lack of fit test (Table S1†). At the concentration ranges of 10–1700 pM for G–T and 20–1200 for A–C, the regression and linearity are acceptable. The values of Fisher ratio (1510.475 and 689.642 for G–T and A–C, respectively) for regression were higher than the critical F-value (4.381 and 4.492) at α = 0.05, which confirmed that the responses are significantly correlated to the concentrations. Moreover, the values of Fisher ratio (namely, 1.488 and 1.869 for G–T and A–C, respectively) for the lack of fit are lower than the critical F-value (2.958 and 3.259) at α = 0.05, and thus the linearity is acceptable as well.

Fig. 6 DPV of oxidation of DABA and AgNPs at different concentrations of G–T (A) and A–C mismatch targets (B) on the GR–AuNPs/GCE with calibration curves (insets).

4. Conclusion

A signal amplification strategy for the improvement of our previously reported protocol for electrochemical SNPs genotyping using M-NPs¹⁶ by applying a GR–AuNPs/GCE platform is investigated, which exhibits excellent sensitivity and selectivity for G–T and A–C mismatch targets with detection limits as low as 2 and 10 pM, respectively. GR–AuNPs not only offer high surface area and good biocompatibility as an immobilization platform, but also GR with a high electrical conductivity could also enhance the electron transfer between DNA and the electrode. Table 1 shows a comparison between the present protocol and some other previously reported biosensors for the genotyping of SNPs. This comparison demonstrates that the present study on the quantification of all SNPs is promising.

Table 1 Comparison between the present study and other previously reported biosensors for the genotyping of SNPs

Method^a	Technique/type of mismatch^b	Detection limit	Ref.
a ApoNPs, apoferritin nanoparticles; QC, quantum dot.b SWV, square wave voltammetry; QCM, quartz crystal microbalance; SPR, surface plasmon resonance; LSAV, leaky surface acoustic wave; SERS, surface-enhanced Raman spectroscopy; DPV, differential pulse voltammetry.
Monobase-modified AuNPs	SWV/all the SNPs	29.75 pmol	15
G-modified Cd–ApoNPs	SWV/CC mismatches	0.3 pM	12
Monobase-coded CdS	QCM/all the SNPs	—	17
Monobase-modified QDs	SWV/all the SNPs	—	18
Monobase-modified metal-ApoNPs	SWV/all the SNPs	5 pM (in the case of A–G mismatch)	13
Single base extension	SPR/A–C mismatches	100 pM	58
Surface ligation reaction	LSAV/A–G mismatches	2 pM	59
Peptide nucleic acid-based methods	SERS/A–G mismatches	3.4 pM	60
Mismatch binding protein-based	QCM/T–G mismatches	1 nM	61
Mismatch binding protein-based	Fluorescence/all of the SNPs	5 nM (in the case of G–T mismatches)	62
Molecular beacon-based	Amperometry/G–G mismatches	0.1 nM	63
Molecular beacon-based	Fluorescence	40 pM	64
Exonuclease based	Impedance/C–C	42 pM	65
Monobase-conjugate nanoparticles on gold electrode	DPV/all the SNPs	5 pM (in the case of G–T mismatch)	16
Modified nanoparticles on gold nanoparticles–graphene	DPV/all the SNPs	2 and 10 pM (for G–T and A–C mismatch targets)	Present study

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Acknowledgements

We gratefully acknowledge the support of the Esfahan University Research Council to this work.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03794h

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