Sequence-specific detection of either genetically or pathogenically associated nucleic acids has become increasingly important for applications including point-of-care diagnostics, antiterrorism, environmental monitoring and forensic analysis. It is highly desirable to develop DNA detection methods with high sensitivity and selectivity, as well as speed, which has motivated the development of electrochemical DNA biosensors. In electrochemical biosensor, the electrocatalytic properties are strongly related to the microstructure and surface chemistry of biosensors. Compared with traditional carbon materials, GN has very high electrical conductivity, large surface area, profuse interlayer structures and abounding functional groups involved, which was beneficial to the construction of novel electrochemical interface. Also, biocompatible GN not only facilitates bimolecular binding but also accelerates electron transfer, and thus amplifies electrochemical detection signal. Novel electrode nanomaterial, various electrochemical technologies as well as signal amplification strategies open new opportunities for highly sensitive detection of nucleic acids.
3.1 Direct Electrochemical Oxidation of DNA and DNA Damage
Among the various methods for DNA electrochemical analysis, the direct oxidation of DNA is the simplest, which can also provide rapid and sensitive detection of single nucleotide polymorphisms (SNPs) 48–50. Most carbon-based electrodes suffer from problems such as narrow electrochemical potential window, slow electron transfer kinetics, and/or high background current, precluding distinct detection of individual bases in intact DNA by voltammetric sensing. Until now, only electron cyclotron resonance of nanocarbon films 49 and GN-based materials 35 could realize the simultaneous detection of all the four DNA bases in DNA without a prehydrolysis step by the direct oxidation. However, the oxidation of the individual bases in DNA is much more difficult than the oxidation of free DNA bases. Many researches on the detection of bases in DNA were reported with the help of a prehydrolysis step to release the bases into their free states 51.
Our group for the first time reported the simultaneous electrochemical sensing of all four DNA bases at GN-based electrode (Figure 2A) 35. Without the need of a prehydrolysis step, the four bases in both ssDNA and dsDNA can be simultaneously detected at physiological pH at the GN electrode, which has higher electrochemical activity than graphite and bare glassy carbon electrodes (Figure 2B,C). Furthermore, electrochemical detection of SNPs was simultaneously realized at physiological pH (Figure 2D,E). The unique physicochemical properties of GN, such as the single-sheet nature, high conductivity, large specific surface area, high density of edge-plane-like defective sites and antifouling properties, induced the accelerating electron transfer and thus enhanced electrochemical activities. Subsequently, several works were reported to construct GN-based electrochemical sensing platform to detect ssDNA and dsDNA via direct oxidation 46, 52–54. Loh et al. prepared the anodized epitaxial GN and systematically investigated the effect of edge plane defects on the heterogeneous charge transfer kinetics 46. Due to a large amount of high edge plane defects involved, anodized GN exhibited a superior biosensing activity for the detection of nucleic acids. The detection limit for dsDNA could reach to 1 µg/mL. In addition, ssDNA, dsDNA as well as SNPs can be differentiated on anodized GN 46. Guanine has the lowest oxidation potential of all DNA bases, thus this base can be more easily detected 55,56. A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide and its metabolite was presented using guanine oxidation 57. In this method, horseradish peroxidase (HRP) and natural dsDNA were alternately assembled on the GN-ionic liquid-Nafion/pyrolytic graphite electrode (PGE). With the guanine signal in DNA as an indicator, the damage of DNA was detected by DPV after the obtained electrode was incubated in the presence of acrylamide and H2O2.
Figure 2. Differential pulse voltammetry (DPV) curves for a mixture of G, A, T, and C (A), ssDNA (B) and dsDNA (C) at GN/GCE (light grey), graphite/GCE (grey), and GCE (black), respectively; concentration for G, A,T, C, ssDNA or dsDNA: 10 µg/mL. Detection of SNPs of oligonucleotides including the sequence from codon 248 of the p53 gene at the GN/GCE: (D) DPV curves of wild-type oligonucleotide 1 and its single base mismatch 2 (GA mutation); (E) DPV curves of wildtype 1 and its single-base mismatch 3 (CT mutation). Concentration for different oligonucleotides: 1 (1 µM), 2 (1 µM) and 3 (1 µM); electrolyte: 0.1 M pH 7.0 PBS (from  with permission).
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Besides the high edge plane defects and functional groups within GN, the morphology and composition of GN-based nanomaterials have the significant effect on the high resolution electrochemical sensing. The reduction in the specific surface area and the embedding of the edge plane defects sites due to the restacking of GN during its processing as a result of the strong van der Waals interactions will decrease the electrochemical activities towards nucleic acids assays. As shown in Figure 3, Akhavan’s group investigated the electrooxidation of DNA at the GN nanowalls electrode 58. The linear dynamic detection range of the GN nanowalls electrode for dsDNA was checked in the wide range of 0.1 fM to 10 mM. The detection limit of the GN nanowalls was estimated as 9.4 zM. The GN nanowalls were efficient in label-free detection of SNPs of 20 zM oligonucleotides. The porous structure with extraordinary edge plane defects for a more efficient heterogeneous electron exchange accounted for the extremely enhanced electrochemical reactivity for DNA detection. Jiao et al. synthesized Au nanoparticles (NPs)/GN composite films on glassy carbon to fabricate the direct electrochemical DNA sensors. Because of the significantly synergistic electrocatalytic effect of GN and Au NPs, synthetic sequence-specific DNA oligonucleotides were successfully detected with high sensitivity and excellent stability and the established immobilization-free biosensor had the ability to discriminate single- or double-base mismatched DNA 59.
Figure 3. (A) SEM images of the GN nanowalls. (B) log-log plot of the current response of the GN nanowalls and GN electrodes to the four bases (G, A, T, and C) of the dsDNA at various concentrations (from  with permission).
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3.2 Sequence-Specific DNA Hybridization Electrochemical Devices
Compared with the direct electrochemical oxidation of DNA, DNA electrochemical devices based on hybridization were regarded to be more efficient because of profuse electrode materials, different electroanalytical principles as well as various signal amplification strategies 60–62. The hybridization between the probe DNA and the target would greatly change the conformation of DNA and these changes could be easily reflected through the electrochemical signal. In addition to excellent conductivity, the anchoring of the DNA probe to GN can be achieved by either covalent grafting or noncovalent π–π stacking readily. Meanwhile, the high specific surface area of GN contributes to the high loading of the DNA probe. These unique properties can be further used to construct different sensing platforms with outstanding performance in DNA electrochemical analysis.
The most common electrochemical strategies for detecting the hybridization of DNA rely on interacting electroactive substances such as a groove binder or intercalating organic compounds (e.g., daunomycin 63,64, methylene blue 65, [Ru(NH3)6]3+) that interact in different ways with ssDNA or dsDNA. Huang et al. constructed GN/polyaniline nanowires modified electrodes 63 and Prussian blue/GN paste electrodes 64, and used them as an advanced electrochemical sensing interface to covalently graft ssDNA probe. Due to the unique synergetic effect, these functional electrodes showed high selectivity and sensitivity towards complementary DNA sequence. DNA/GN/polyaniline nanocomposites were reported as an electrochemical DNA sensing platform to recognize specific DNA hybridization 66. In the presence of target DNA, ssDNA probe noncovalently assembled on GN would release due to the strong and stable binding. The transformation variation could be probed by the redox current changes of [Ru(NH3)6]3+. Besides, some related amperometric DNA sensors based on assembly of GN and DNA-conjugated Au NPs with silver enhancement strategy 67, Au NPs decorated GN-PAMAM dendrimer 65 and GN-supported ferric porphyrin as peroxidase mimic were also reported previously.
It should be noted that multiple signal amplification approaches combining with other functional nanomaterials were also adopted in GN-based amperometric DNA sensors. For example, our group synthesized GN-mesoporous silica-gold nanoparticle hybrids (GSGHs) and utilized this functional material as enhance electrochemical platform to sensitively detect DNA 69, chiral D-vasopressin 18 and adenosine triphosphate 70. GSGHs were first synthesized via a simple wet-chemical process combining self-assembly technique (Figure 4). Then, GSGHs-based layer-by-layer (LBL) sensing interface was constructed through the electrostatic interaction 69. The use of triplex DNA system for amplifying the detection of DNA on the integrated functional interface via combining the strand-displacement DNA polymerization reaction was demonstrated. The present electrochemical detection offers some unique advantages such as ultrahigh sensitivity, simplicity, and feasibility for apparatus miniaturization in analytical tests. Triplex signal amplification for electrochemical DNA biosensing by coupling probe-Au NPsAu modified electrode with enzyme functionalized carbon sphere as tracer was also demonstrated. This novel signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5 aM) with a linear range of 5 orders of magnitude (from 1×10−17 M to 1×10−13 M), and high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA appeared 71.
Figure 4. (A) Schematic procedure for the synthesis of GN-mesoporous silica-gold NPs hybrid nanosheets. (B) Illustration of the procedure for preparing the electrochemical sensing interface. (C) The procedure of strand-displacement DNA polymerization amplification and parallel-motif DNA triplex amplification (from  with permission).
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Recent studies revealed that ss-DNA could form a stable nanocomposite with GN through π–π stacking interactions 72, 73. It would be of particular interest to develop label-free DNA electrochemical impedance sensors using the hybridization event through changes in recognition layer’s physical and chemical properties. Generally, after hybridization with a complementary target, a significant decrease in charge transfer resistance (Rct) value was observed, due to the partial release of the DNA probes from the GN surface during hybridization 74. Pumera’s group employed GN platforms modified with hairpin-shaped DNA (hpDNA) probes for the sensitive detection of SNPs correlated to Alzheimer’s disease 75. As shown in Figure 5, after hybridization with a complementary target, a significant decrease in Rct value was observed, due to the partial release of the hpDNA probes from the GN surface during hybridization. However, the Rct decrease was less significant in the case of hybridization with a mutant and no average Rct variation was observed with the noncomplementary sequence. The present label-free strategy could achieve a linear range between 0.3 pM and 0.3 nM for the detection of the wild-type target. Furthermore, they also compared different GN materials (graphite oxide, graphene oxide (GO), thermally reduced GO (TR-GO) and electrochemically reduced GO (ER-GO)) platforms for the impedimetric detection of DNA hybridization by immobilizing DNA probes by physical adsorption or covalent binding to GN 76, 77. It is found that π–π-stacked DNA probes on GO provided the best ability to discriminate a complementary sequence from one carrying a SNPs 76, while the best performance, in terms of sensitivity and reproducibility, was achieved with ER-GO platform through covalent grafting approach 77. Loh et al. compared the results obtained by immobilizing DNA probes by covalent grafting or noncovalent π–π stacking on an anodized epitaxial GN (EG) platform 52. Compared with noncovalent π–π stacking DNA on anodized EG, label-free DNA detection by electrochemical impedance spectroscopy (EIS) based on covalently bound DNA could provide a larger detection range and a more sensitive response. Different from the partial release of DNA probes from the GN surface during hybridization, they attributed the decresed Rct upon addition of target DNA to the better conductivity and more effective electron transfer induced by the formation of dsDNA on GN surface.
Figure 5. A schematic of the protocol for the GN-based EIS sensing platform and Nyquist plots showing −Zi vs. Zr for the GN surface, hpDNA, complementary target, 1-mismatch target, and a negative control with a noncomplementary sequence (NC) (concentration of the DNA probes was 1×10−5 M; concentration of the DNA target was 3×10−8 M). All measurements were performed in 0.1 M PBS buffer solution containing 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (from  with permission).
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Niu et al. also developed a serial of label-free electrochemical impedance sensing of DNA hybridization based on functionalized GN 39, 61, 78–79. It is pointed out that the increase in Rct values was observed in the presence of a target DNA on the functionalized GN sensing platform. Namely, double-helix structure formed after hybridization stayed on functionalized GN platform with the assistance of hydrophobic, electrostatic/hydrogen bonding interactions and weak π–π stacking. Notable example was that they successfully synthesized N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide/GN (PDI/GN) via π–π stacking interaction 61. Electrostatic interaction between PDI’s positively charged imidazole rings and negatively charged phosphate backbones of ssDNA facilitates ssDNA immobilization. Impedance value of the PDI/GN platform increased after probe DNA immobilization and hybridization to its complementary sequences (Figure 6). The conserved sequence of the pol gene of HIV-1 was satisfactorily detected with detection limit of 5.5×10−13 M and good selectivity. Positively charged Au/GN nanocomposites were also used to design the label-free DNA sensor based on increasing impedance value in the presence of target DNA 80. Similar with this positively charged GN nanomaterials, the dsDNA could also lie on the electrochemical reduced GO (ERGO) surface to form a stable ERGO-dsDNA complex after hybridization. We speculate that the change in impedance value observed largely depends on the GN-based electrode interface (charge loaded and functional groups) and the intrinsic attributes of dsDNA formed. Although the detailed mechanism is not fully understood, it is clear that the EIS can be used for label-free detection of target DNA with high sensitivity.
Combining the high sensitivity of the EIS technique with an enzyme-assisted target recycling signal to detect target-gene sequences was also reported. Chen et al. reported label-free genosensing using GN/Au NPs nanocomposite on which a hairpin DNA probe was immobilized 81. The presence of exonuclease III leads to direct recycling and reuse of the target DNA, which in turn results in substantial signal amplification for highly sensitive, label-free impedimetric detection of specific DNA sequences. This unique property of Exo III makes the GN-based EIS sensor hold great potential for highly sensitive, selective and simple detection of a wide range of target DNA sequences.