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Keywords:

  • Graphene;
  • Electrochemical sensors;
  • Nucleic acids;
  • Proteins;
  • Cancer cells

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

In recent years graphene (GN) received widespread attention owing to its extraordinary physical and chemical properties. Lately, considerate efforts have been devoted to explore potential applications of GN in life science, especially in disease-related diagnostics and detection. Especially, the coupling of electrochemical devices with the GN offers an excellent platform to realize the diagnostics and detection of nucleic acid, protein and cancer cells with high performance. This review focuses on the rising progress on GN-based nanomaterials as advanced electrochemical sensing devices for the detection of the above-mentioned targets. Future challenges and perspectives in this rapidly developing field are also discussed.


1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Graphene (GN), a two-dimensional (2D) crystal of sp2-hybridized carbon atoms arranged in six-membered rings, has attracted tremendous attention and research interest since its discovery in 2004 12. This exciting new material has quickly sparked great interests across many disciplines, including nanoelectronics, energy storage and conversion, and bioscience/biotechnologies because of its unique physicochemical properties: the quantum hall effect, high carrier mobility at room temperature (200 000 cm2 V−1 s−1), high Young’s modulus (∼1100 GPa), good optical transparency (∼97.7 %), high surface area (theoretically 2630 m2/g for single-layer GN), excellent thermal conductivity and electric conductivity, and strong mechanical strength 34. Among them, the emergence of GN provides an excellent electrode material. Great efforts have been made to utilize GN-based nanomaterials as promising electrode materials with high performance 57. In addition, a number of approaches to obtain high-quality GN have been explored, further holding great promise for the wide application of GN-based nanomaterials 3, 89.

Bioanalysis plays an ever-increasing role in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, various cancers, and clinical medicines 10, 11. For example, many disorders, such as Alzheimer’s disease and various cancers, are closely related with DNA damage. The most efficient strategy to halt cancer’s progress is to develop new diagnostic tools that allow potential biomarkers disease to be detected at early stage 12. Sensitive and selective detection of nucleic acids, proteins and cancer cells is in urgent need due to their important roles in human bodies. Over the past decades we have witnessed a tremendous amount of activity in this area. Various types of optical, resonant, thermal, electrochemical, FET-based biosensors and diagnosis platforms are used for disease-related diagnosis and detection 1317. Electrochemical biosensors own many advantages, such as being easy to operate, economic, sensitive, and suitable for automation, miniaturization and field analysis 1820. Significantly, extensive research on construction of functional electrode materials, coupled with numerous electrochemical methods, has opened the door to widespread applications of electrochemical devices.

Based on the unique properties, GN was proved an ideal nanomaterial for the preparation of electrochemical biosensors. Papakonstantinou et al. first demonstrated the example of the use of GN-based nanomaterials for electrochemical sensing. They found that GN exhibited faster electron-transfer kinetics and had a high performance for the simultaneous detection of dopamine, ascorbic acid and uric acid 21. Subsequently, great contributions were made to rationally design GN-based nanomaterials and use them as advanced electrode nanomaterials in electrochemical biosensors. To date, great efforts were made to develop GN-based small molecules sensors, such as H2O2 22, 23, N2H4 24, dopamine 25, 26, NADH 27, 28, and so on 2931. GN-based nanomaterials are also expected to promote the electron transfer between electrode substrates and enzymes to fabricate electrochemical enzyme sensing platform 3234. Notable contribution was that our group systematically demonstrated the application of GN for the preparation of an advanced electrochemical sensing and biosensing platform, which exhibited high electrosensing ability towards different targets, such as small molecules and biomolecules 35. Furthermore, an enormous array of works on the electrochemical detection of DNA, protein and cancer cell with high sensitivity was reported previously 3639. These successful demonstrations strongly confirmed that the distinct electronic features and enhanced electrochemical performance could endow GN-based nanomaterials with multiple functions, and might offer an effective platform for the development of new kinds of nanostructured electrochemical sensing devices. In this review, we stress on the recent advances in GN-based nanomaterials as advanced electrochemical sensing devices for the detection of nucleic acid, protein and cancer cell (Figure 1). Combining with the novel GN-based nanomaterials, the attractive electrochemical devices are extremely promising for improving the efficiency of disease-related diagnostics and detection.

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Figure 1. Representation of GN-based electrochemical sensors in DNA, protein and cancer cell detection.

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2 GN-Based Electrochemical Sensing Interface

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Carbon-based nanostructures such as fullerene, carbon nanotubes (CNTs), and mesoporous carbons have been extensively used in electrochemistry because of their low price, suitable electrocatalytic activity for a variety of redox reactions, a broad potential window, and relatively inert electrochemistry 4042. Recently, the emergence of the GN induces an explosion of research in the use of GN-based nanomaterials for constructing high-performance electrochemical sensing devices based on different sensing strategies 43, 44. Due to the two dimensional nature of GN, heterogeneous electron transfer with redox species can take place on the edges of GN, while heterogeneous electron transfer from the plane of a GN sheet is almost nil 45. Recent studies revealed that edges plane defects and oxygen-containing groups within GN induced by oxidation play a significant role in facilitating the heterogeneous electron transfer 46. However, the origin of these intriguing properties remains an open question and detailed mechanism is still underway. Still, there is evidence that GN and its derivatives can exhibit good electrochemical performance compared with other electrodes such as glassy carbon, graphite, or even CNTs 29, 35. On the one hand, a number of approaches to obtain high-quality GN have been explored, making the GN a promising nanomaterial in electrochemical biosensor 47. On the other hand, rational synthesis of GN-based hybrids with different nanostructures will no doubt optimize the construction of the electrochemical interface, thus enhancing the electrochemical performances and expanding the potential application of GN nanomaterials. GN-based functional biosensing interface can not only produce a synergic effect among catalytic activity, conductivity and biocompatibility to accelerate the signal transduction, but also provide amplified recognition events by high loading of signal tags, leading to a highly sensitive and specific biosensing.

3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

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) 4850. 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, 5254. 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.

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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 (G[RIGHTWARDS ARROW]A mutation); (E) DPV curves of wildtype 1 and its single-base mismatch 3 (C[RIGHTWARDS ARROW]T mutation). Concentration for different oligonucleotides: 1 (1 µM), 2 (1 µM) and 3 (1 µM); electrolyte: 0.1 M pH 7.0 PBS (from [35] 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.

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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 [58] 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 6062. 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+[66]) 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[68] 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 NPs[BOND]Au 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.

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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 [69] 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.

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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 [72] 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, 7879. 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.

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Figure 6. Schematic representation of DNA hybridization on PDI/GN platform (from [61] with permission).

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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.

4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Immunoassays, based on the specific reaction of the antigen[BOND]antibody recognition, have been a major analytical tool and gained increasing attention in clinical, food, environmental-based bioanalysis 11, 82. Electrochemical methods appear as the most promising alternative to ELISA (enzyme linked immunosorbent assay) approaches. Of note, a sandwich-type protocol is adopted to prepare the electrochemical immunosensor. Typical procedures include the immobilization of a recognition element (i.e. antigen or antibody) on the electrode surface, the specific binding of antigen and the introduction of a secondary enzyme-labeled antibody. It is clear that the immobilization of the recognition element on the electrode interface and the rational design of signal tags play crucial role in developing the advanced electrochemical immunosensor. Biocompatible GN as an excellent sensing platform not only presents an abundant domain for bimolecular binding but also is favorable for fast electron-transfer kinetics and further signal amplification in electrochemical immunosensor. Up to now, there are many successful demonstrations in constructing GN-based electrochemical immunosensors for detection of a large series of targets with high sensitivity, such as cancer biomarkers 83, 84, proteins 85, 86, virus 87, and so on 88, 89. The recent advances in electrochemical immunosensors based on GN nanomaterials were summarized in Table 1.

Table 1. Summary of electrochemical immunosensors based on GN nanomaterials. CA: chronoamperometry; ASV: anodic stripping voltammetry; PPD: poly(ophenylenediamine); MB: magnetic bead; MCPE: magnetic carbon paste electrode; TH: thionine; BSA: bovine serum albumin; GOD: glucose oxidase; SPCE: screen-printed carbon electrodes; SWV: square wave voltammetry; TCPP: meso-tetra(4-carboxyphenyl)porphyrin; cTnI: human cardiopathy biomarkers cardiac troponin I; FABP: human heart-type fatty-acid-binding protein; hCG: human chorionic gonadotrophin; APS: 3-aminopropyltriethoxysilane; PSA: prostate specific antigen; ALVs-J: subgroup Jofavianleukosis virus; DA: dopamine; FC: ferrocene carboxylic acid; SCC-Ag: squamous cell carcinoma antigen; PPA: 3-phosphonopropinic acid; LDHs: layered double hydroxides.
GN-based electrochemical sensorsDetection methodTargetLiner rangeDetection limitReference
anti-CEA/Ag NPs/PPD/MB/MCPE coupled with HRP-anti-CEA/Au NPs-GO signal tagsDPVCEA0.01–40 ng/mL0.001 ng/mL[94]
PVP-GS/TH/GCE using SBA-15/HRP/Ab2/IL as labelsDPVBRCA10.01–15 ng/mL4.86 pg/mL[80]
ZnO NPs@GN-GOD-Ab2/ CEA/BSA/Ab1/AuNPs@GN/GCEECLCEA0.01–80 ng/mL3.3 pg/mL[101]
Ab1/AuNPs/SPCE combined with HRP-Ab2-GOSWVP53 phosphorylation0.02–2 nM0.01 nM[91]
HRP-Ab2-CNSs/AFP/Ab1/GN/SPCESWVAFP0.05–6 ng/mL0.02 ng/mL[93]
GN nanoribbons/GCE using anti-cTnI-TiP-Cd2+ and anti-FABP-TiP-Zn2+ bioconjugates as signal tagsSWVcTnI and FABPcTnI: 0.05 pg/mL–50 ng/mL FABP: 0.05 pg/mL–50 ng/mLcTnI: 1 fg/Ml FABP: 3 fg/ml[97]
GN-CdS/agarose/APS/BSA/Ab/GCEECLAFP0.0005–50 pg/mL0.2 fg/mL[103]
Au NPs/PEI-PTCA /GCE using bio-AP/SA/anti-fPSA(PSA)/Au@PBNPs(Au@NiNPs)/O-GN as tracersDPVfPSA(PSA)fPSA(PSA): 0.02–10 (0.01–50) ng/mLfPSA(PSA): 6.7 (3.4) pg/mL[102]
DA-Fe3O4-FC-Ab2/PSA/Ab1/GN/GCESWVPSA0.01–40 ng/mL2 pg/mL[95]
Ab/Au/CdSe-GN/GCEECLhuman IgG0.02–2000 pg/mL0.005 pg/mL[85]
HRP-SCC-Ab/Au/GN/SCC-Ag/ Au/TH/NiCo2O4/Au electrodeDPVSCC-Ag2.5 pg/mL–15 ng/mL1.0 pg/mL[104]
PSA /Ab2 – Si/QDs/TH – GA – Ab1GN@Fe3O4/ITOECLPSA0.003–50 ng/mL0.72 pg/mL[96]
HRP – Ab2/AuNPs/PDDA-GO bioconjugate/ IgG/BSA/Ab1/AuNPs/PDDA-GN/GCEDPVhuman IgG0.1–200 ng/mL0.05 ng/mL[105]
Ab2-HRP/ IgG /Ab1/GN – MWCT/GCECVhuman IgG1–500 ng/mL0.2 ng /mL[90]
HRPanti-AFP/AuNP/GN-doped chitosan /PTH/GCEDPVAFP1.0–10 ng/mL0.7 ng/ml[83]
HRP-Ab2/Au/TH/MCM-41 bioconjugate hCG/Ab1/Au/MWCNTs/GN/GCEDPVhCG0.005–500 mIU/mL0.0026 mIU/mL[100]
Ab2-GN-QDs/antigen/Ab1/PPA/ITOASVepithelial cell adhesion molecule (EpCAM) antigen100 fg/mL[92]
HRP-Ab2/Fc-LDHs@Fe3O4/ALVs-J/GN-LDHs/GCEDPVALVs-J102.32–105.50 TCID50/mL180 TCID50/mL[87]
GN nanoribbons-BTX-2-BSA conjugates/Fe3O4-Ab/MPCESWVbrevetoxinB (BTX-2)1.0 pg/mL–10 ng/mL1.0 pg/mL[106]
Ab-HRP-AuTi conjugates/Ab/AuAg-GN/Th/GCEDPVAFP0.001–200 ng/mL0.5 pg/mL[107]
GOD- conjugated gold – silver hollow microspheres/Ab/CEA /Ab/Au/PB-GN/GCEDPVCEA0.005–50 ng/mL1.0 pg/mL[84]
Au hollow microspheres-Ab/Ag/Ab-Fe3O4-GN/ITODPVCEA and AFPAFP: 0.01–200 ng/mL CEA:0.01–80AFP(CEA): 1.0 pg/mL[99]
Au-Fe3O4-Ab2/ antigen /Ab1-GN-GCECAPSA0.01–10 ng/mL5 pg/mL[108]
Au-MSN-HRP-Ab2/ norethisterone/Ab1-TH-GN-GCECVnorethisterone0.01–10 ng/mL3.58 pg/mL[88]
Gox-Au nanorods -Ab2/PSA/Ab1/GN-chitosan/GCEECLPSA10 pg/mL–8 ng/mL8 pg/mL[98]
GN-TH-HRP-Ab2/PSA/Ab1-GN/GCECAPSA0.002–10 ng/mL1 pg/mL[38]
GN-modified SPCEDPVβ-lactoglobulin0.001–100 ng/mL0.85 pg/mL[109]
TCPP/GN/GCEEIScyclin A20.32 pM[89]
ERGO/ITOCVmouse IgG100 fg/mL[86]

Tang et al. developed a new electrochemical immunosensing protocol for sensitive detection of α-fetoprotein (AFP) in human serum by means of immobilization of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) onto GN and Au-functionalized biomimetic interfaces 83. The low-toxic and high-conductive GN complex provided a large capacity for nanoparticulate immobilization and a facile pathway for electron transfer. LBL assembly of GN and CNTs for sensitive electrochemical immunoassay human IgG was reported by Li et al. 90. The assembled composite film significantly improved the interfacial electron transfer rate compared with that of GN or CNTs modified electrode due to 3D porous structure interface. This electrochemical immunosensor exhibited excellent selectivity, stability and reproducibility, and can be used to accurately detect IgG concentration in human serum samples. Zhu’s group proposed a new strategy for the fabrication of an advanced electrochemiluminescence (ECL) immunosensor by using PDDA-GN 85. As shown in Figure 7, CdSe quantum dots (QDs) were successfully attached to PDDA-GN through electrostatic interactions, and the obtained PDDA-GN-CdSe composites were used to construct an ECL immunosensor via LBL assembly. After two successive steps of amplification via the conjugation of PDDA and Au NPs in the film, high ECL intensity is observed. The ECL immunosensor has an extremely sensitive response to HIgG in a linear range of 0.02–2000 pg/mL with a detection limit of 0.005 pg/mL. Also, functionalized GN as a nanocarrier in multienzyme labeling amplification strategies for construct ultrasensitive electrochemical immunosensors were demonstrated in previously References 91, 92.

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Figure 7. (A) Schematic representation of the preparation procedure of PDDA-GN-CdSe composites, including the oxidation of graphite (gray blocks) to GO with abundant oxygen functionalities, the in situ reduction of GO in the presence of PDDA to obtain positively charged PDDA-GN colloids, and the preparation of PDDA-GN-CdSe composites via electrostatic interactions under sonication. (B) Schematic illustration of the stepwise immunosensor fabrication process, including the formation of PDDA-GN-CdSe composite film on the Au electrode, the linkage of PDDA to the film, the conjugation of gold NPs (GNPs) to PDDA, the immobilization of antibody (Ab) on the electrode via GNPs, and the specific immunoreaction (from [85] with permission).

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Remarkably, the introduction of GN coupled with other nanomaterials is not only beneficial to the immobilization of a recognition element but also can significantly increase the enzyme (electroactive species) loading toward a sandwich immunological reaction event, which further enhance the detection sensitivity due to dual signal amplification platform. Lin et al. reported sensitive immunosensor for AFP based on dual signal amplification strategy of GN and multienzyme functionalized carbon nanospheres (CNSs). This novel biosensors possessed the following advantages: (1) CNSs as the enzyme-loading carrier can load many enzyme molecules on each CNSs. (2) GN can provide a high density of primary antibodies because of their high surface area. The resulting immunosensor possessed high sensitivity, good reproducibility, and cost-effective analytical performance 93. Wei and co-workers developed an immunosensor using the SBA-15 nanoparticles and ionic liquid (IL) as labels for BRCA1 detection (Figure 8) 80. Horseradish peroxidase (HRP) was entrapped in the pores of amino-group functionalized SBA-15 and the secondary antibody (Ab2) combined with SBA-15 by covalent bond. GN-based electrode interface together with SBA-15/HRP/Ab2/IL as labels displayed a linear response for detection BRCA1 within a wide range (0.01–15 ng/mL). The proposed biosensor shows low detection limit (4.86 pg/mL), good reproducibility, selectivity and acceptable stability. Moreover, combined with diverse nanomaterials (magnetic bead 9496, TiP 97, Au nanorods 98, noble metal hollow microspheres 84, 99, porous nanomaterials 88, 100, etc. 87, 101) as enhancers, great efforts were made to provide a new opportunity for the development of high-performance electrochemical immunosensors. Furthermore, simultaneous multianalyte determination via distinguishable signal tags could also be easily realized on GN-based electrochemical immunosensors 97, 99, 102. Notable contribution was that Knopp et al. developed a novel highly sensitive multiplexed electrochemical immunoassay for simultaneous detection of AFP and carcinoembryonic (CEA) using biofunctionalized magnetic GN as immunosensing probes and multifunctional Au hollow microspheres as distinguishable signal tags (Figure 9) 96. It is revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01–200 ng/mL for AFP and 0.01–80 ng/mL for CEA with low detection limits for both analytes at 1.0 pg/mL. This approach does not require sophisticated fabrication and is well-suited for high-throughput biomedical sensing and application to other areas.

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Figure 8. Schematic representation of the fabrication of the immunosensor (from [80] with permission).

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Figure 9. Schematic illustration of the multiplexed electrochemical immunoassay protocol and the measurement principle of the sandwich immunoassay (from [96] with permission).

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Besides, using high binding affinity and specificity between aptamer and enzyme, electrochemical aptasensors based on GN nanomaterials have offered a new generation of biosensing platforms because of their high binding affinity and specificity to a broad range of targets from small inorganic and organic substances to proteins and cells 60, 110111. Our group for the first time presented GN as a new substrate for the development of highly sensitive surface plasmon resonance (SPR) and electrochemical sensors for a thrombin assay. As shown in Figure 10, the α-thrombin aptamer (TBA) was adsorbed onto the GN layer through the strong noncovalent binding of GN with the nucleobases. Upon addition of the target molecule, TBA could be released from the SPR sensing surface and an obvious SPR angle decrease could be observed. The detection range of the proposed aptasensor could extend up to 200 nM, with the detection limit down to 0.03 nM 37. Yuan’s group successfully fabricated a highly sensitive and selective aptasensor for thrombin detection with use of hemin/G-quadruplex system and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. The proposed aptasensor exhibited extraordinary electrochemical biocatalysis in the presence of nickel hexacyanoferrates nanoparticles (NiHCFNPs) toward H2O2, which largely increased the sensitivity of proposed aptasensor. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained 112.

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Figure 10. Fabrication of the sensing interface and the detection of a-thrombin. The characterization of GN, preparation of the p-Au film and assembly process of GN and TBA onto the p-Au film (from [37] with permission).

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5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Detecting cells, as well as rare pathological bacteria and pathogens, is of obviously clinical significance. GN-based nanomaterials have been developed for constructing cell-related electrochemical devices and significant progress has been made for the detection of cells with good sensitivity and selectivity. He et al. proposed a novel biocompatible film assembled by combining GO and poly-L-lysine(PLL), which showed an improved immobilization capacity for living cells and a good biocompatibility for preserving the activity of the immobilized living cells 113. Electrochemical impedance method for the detection of leukemia K562 cells based on this biocompatible film was facially realized with high sensitivity. The functionalization of GN and the synthesis of the GN hybrids were focused on to anchor the recognition element, making the sensor specific for target cells 114117. Qu and colleagues developed functionalized GN-based electrochemical aptasensor for detecting cancer cells by combing the high binding affinity and specificity of clinical trial II aptamer AS1411 to nucleolin, which is overexpressed on the surface of cancer cells 114. As shown in Figure 11, nanoscale anchorage substrate was constructed through the covalent linking between functionalized GN and NH2-modified AS1411. The resultant aptasensor could distinguish cancer cells and normal ones and detect as low as one thousand cells. Folate receptors (FR) are the protein receptors on the surface of cell membrane, which are usually overexpressed on some tumor cells, and hence folic Acid (FA) can specifically target FR with high affinity. LBL assembly of carboxymethyl chitosan-GN functionalized with polyethyleneimine and FA was reported by Zhu et al. for label-free electrochemical detection of HL-60 cells with high stability and biocompatibility 115. In the meantime, an advanced photoelectrochemical cytosensor based on GN[BOND]CdS nanocomposites followed by LBL assembly process were also demonstrated to selective detection of Hela cells 117. Besides, Liu et al. proposed the new sensing strategy for sensitive detection of MCF-7 cells based on ECL resonance energy transfer (ERET) from bis(2,20-bipyridine)-(5-aminophenanthroline)ruthenium(II) (Ru1) to GO. The presented method could respond at concentrations as low as 30 cancer cells per mL 118.

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Figure 11. Schematic representation of the reusable aptamer/GN-based aptasensor. The sensor is constructed based on GN-modified electrode and the first clinical trials II used aptamer, AS1411. AS1411 and its complementary DNA are used as a nanoscale anchorage substrate to capture/release cells (from [114] with permission).

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Reactive oxygen species (ROS) produced in various physiological processes are a series of potentially hazardous species in some systems. ROS are also considered as biochemical mediators in cellular pathology and can be used as an early indicator for cytotoxic events and cellular disorders. Li et al. constructed a smart multifunctional biointerface by growing human cells on layered GN[BOND]artificial peroxidase[BOND]protein (Figure 12) 119. Among them, GN provides the platform to construct the biointerface with dimensional compatibility for good growth of human cells and excellent electrical conductivity for electrical detection, while incorporating artificial peroxidase and extracellular matrix protein achieves good selectivity and enhances cell adhesion/growth capability, respectively. In situ selective and quantitative extracellular H2O2 detection has been realized on this smart multifunctional biointerface 119. A new type of flexible electrochemical sensor fabricated by depositing high-density Pt NPs on freestanding GN paper carrying MnO2 nanowire networks was also reported. This triple-component design offers new possibilities to sensitively monitor H2O2 secretion by live cells by virtue of the mechanical and electrical properties of GN paper, the large surface area of MnO2 networks, and the catalytic activity of well-dispersed and small sized Pt NPs 120. Cai and co-workers used N-doped GN as enhanced electrochemical sensing platform for measuring the dynamic process of H2O2 release from living cells. This approach could be potentially useful in the study of downstream biological effects of various stimuli in physiology and pathology 121.

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Figure 12. (A) Scheme showing one path for PMA-triggered H2O2 production from a human cell. (B) Amperometric responses obtained at control 1 (ITO/(GN[BOND]AP[BOND]laminin) 10 without cells under PMA injection), control 2 (ITO/(GN[BOND]AP[BOND]laminin) 10 with cultured cells under injection of DMSO, the solvent for PMA), and ITO/(GN[BOND]AP[BOND]laminin) 10 with cultured cells under PMA (5 µg/mL), followed by catalase injection (500 U m−1 ). (C) The corresponding current responses obtained from amperometric curves shown in (B) (from [119] with permission).

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Aside from H2O2, real-time detection of other biointeresting molecules released from living cells could be easily realized at GN-based nanomaterials. For example, a smart functional biomimetic film sensor by covalently bonding RGD-peptide on GN surface was constructed to significantly boost cell-adhesion and growth for real-time electrochemical detection of nitric oxide molecule released from attached human cells under drug stimulations 122. Xu et al. for the first time reported a facile and mild strategy to synthesize K-modified GN, which exhibited excellent electrocatalytic activity toward the oxidation of equation image. Furthermore, the K-modified GN had excellent analytical performance and can be successfully applied in the determination of equation image released from liver cancer and leukemia cells and shows good application potential in biological systems 123. These research advances indicated that these smart nanostructured GN-based functional nanomaterials provide a powerful and reliable platform to the real-time study of biointeresting molecules released from living cells, thus rendering potential broad applications in neuroscience, screening drug therapy effect, and live-cell assays.

6 Conclusions and Perspectives

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

The need for ultrasensitive bioassays has made the functional nanomaterials one of the hottest fields of research. The outstanding electrical properties of carbon nanomaterials make them the ideal electrochemical biosensing platform. Compared to other carbon-based nanomaterials such as carbon nanotube and mesoporous carbon, GN is highly amenable to fabrication with massive production. Besides, high electrical conductivity, large surface area, profuse interlayer structures and abounding functional groups involved are more favorable for the construction of the high-performance electrode materials, thus greatly improving the sensitivity and reproducibility for various targets.

This review highlighted recent important advances in the design of GN-based nanomaterials for application in electrochemical bisensors for the diagnostics and detection of nucleic acid, protein and cell with high performance. The unique chemical and physical properties endow GN nanomaterials with excellent electrochemical signal transduction abilities for design of a new generation of biosensing devices and application in the bioassay. Significantly, the development of label-free technologies based on GN nanomaterials further promotes the electrochemical bisensors in this field. Despite these important achievements, the merging of GN and electrochemical analytical devices are still in its infancy, with many challenges and opportunities remaining. To this end, the following aspects are awaiting to achieve: (1) New methods for obtaining high-quality GN and GN-based hybrids for the construction of highly sensitive electrochemical sensors are highly desirable. Creating abundant edge plane defects and other defects within GN nanomaterials and making full use of them are crucial for the enhanced heterogeneous electron transfer, thus providing the excellent platform in high resolution electrochemical sensing. Furthermore, due to the synergistic contribution of two or more functional components, appropriate designs of GN-based nanomaterials can exhibit the beneficial properties of each parent constituent and produce the improved performance. Considerable efforts are needed to further improve the fabrication methods to make well-defined structures so that 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 will be largely avoided. (2) Due to the diverse properties of different nanomaterials, utilizing other nanomaterials coupled with GN along with different signal amplification strategies can significantly enhance the good qualities, which lead to a highly sensitive and specific biosensing. (3) The coupling of various electrochemical methods with GN offers a unique multiplexing capability for simultaneous measurements of multiple diseased-related targets, offering more convenient, portable and integrative detection equipments. (4) Considerable work must still be done to assess, and optimize, the biocompatibility of GN-based nanomaterials, and to determine any possible toxicity and health risks, realizing the miniaturization and production of GN biosensors as diagnostic devices. With the demand in life sciences and clinical diagnosis, the utilization of GN nanomaterials in this area are thus extremely promising for improvement of the efficiency for diagnostic testing and therapy monitoring, and for point-of-care diagnostic devices.

Biographical Information

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Chengzhou Zhu graduated with a B. S. degree in chemistry from Shandong Normal University (P. R. China) in 2007. Then, he moved to the Changchun Institute of Applied Chemistry as a PhD student under the supervision of Prof. Shaojun Dong, majoring in analytical chemistry and material chemistry. He has co-authored over 30 peer-reviewed publications. His scientific interests focus on carbon and metal nanomaterials for electrochemical applications.

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Biographical Information

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 GN-Based Electrochemical Sensing Interface
  5. 3 GN-Based Nanomaterial for Electrochemical Nucleic Acids Assays
  6. 4 GN-Based Nanomaterials for Electrochemical Immunoassays and Protein Detection
  7. 5 Cell-Related Electrochemical Sensors Based on GN Nanomaterials
  8. 6 Conclusions and Perspectives
  9. Acknowledgements
  10. Biographical Information
  11. Biographical Information

Shaojun Dong is Professor of Chemistry at the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. She is a Member of the Academy of Sciences of the Developing World since 1999. She has been on the Editorial and Advisory Board of six international journals: Chem. Commun., Biosens. Bioelectron., Electrochem. Commun., Sensors, Bioelectrochemistry, and Talanta. Her research interests concentrate in electrochemistry with interdisciplinary fields, such as chemically modified electrodes, nanomaterials and nanotechnology, bioelectrochemistry spectroelectrochemistry and biofuel cells. She has published over 900 papers in peer-reviewed international journals, the citation by others exceeds 25 000, with an h-index of 71.

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