Review—Recent Progress in the Graphene-Based Electrochemical Sensors and Biosensors

In this review we shortly discuss about the graphene and graphene-based materials synthesis and present the recent year’s research progress (2017 − 2019) in the enhancement of the analytical performance of sensors and biosensors. Particularly, we covered a very broad range of graphene-based electrochemical sensors and biosensors for the detection of glucose, cholesterol, dopamine (DA), ascorbic acid (AA), uric acid (UA), bisphenol A (BPA), cancer biomarkers and heavy metal ions. We believe that the discussed subjects are useful and may be used as an instruction guide for the future developments in the ﬁeld of graphene and graphene-based materials for sensors and biosensors. 4.5 μ A/nM. Their studies indicate that the new nanocomposite rep-resents a favorable material for the development of an inexpensive and efﬁcient sensor for Cd 2 + detection. A porous graphene oxide-polypyrrole polymer nanocomposite modiﬁed sensor for electrochem- ical trace analysis of Cd (II) 112 was also developed. The simultaneous detection of Cd 2 + and Pb 2 + was realized with several electrochemical sensors based on nitrogen-doped quan- tum dots graphene oxide hybrid; 113 multilayer graphene paste electrode (mixture of graphene ﬂakes and mineral oil) modiﬁed with activated carbon; 114 graphene nanosheets with hierarchical MgFe-layered double hydroxide (LDH) on the surface; 115 graphene oxide, κ -carrageenan and L-cysteine; 116 a composite ﬁlm of reduced graphene oxide/carboxylation multi-walled carbon nanotubes/gold nanoparticle hybrid material; 117 a ﬂexible Au substrate with micro-patterned reduced graphene oxide and a carbon nanotube composite. 118 ity over time epitaxial graphene oxide was used as a sensitive sensor for the rapid and simultaneous detection of Pb 2 + and Hg 2 + . 121 The limit of detection of the modiﬁed electrode toward Pb 2 and Hg 2 was 0.2 ng/mL and 1 ng/mL respectively, in the linear range of 1 ∼ 1000 ng/mL(for Pb 2 + ) and 1 ∼ 1000 ng/mL (for Hg 2 + ). The proposed modiﬁed electrode may used for qualitative and quantitative detection of heavy metal ions in various real samples.

A considerable progress has been made in the synthesis, processing, characterization and applications of nanomaterials used to design electrochemical sensors and biosensors with considerably enhanced sensitivity. Most important, (bio)sensors have to be specific, reusable and to have high sensitivity toward specific analyte. A variety of nanomaterials with well-controlled physicochemical properties 1-3 are used to modify electrodes in order to improve their analytical performances. Among them, graphene has drawn increasing attention due to its remarkable physical properties (strong mechanical strength, 4 large surface area, excellent thermal, optical and electrical conductivity 5 ). Having unique and convenient morphological properties, graphenebased nanomaterials are very useful for sensing. 6,7 Electrochemical sensors and biosensors with high sensitivity are extremely important not only in biomedical applications, clinical diagnosis but also in environmental protection. 8 This type of sensors have a wide range of sensing capabilities including sugars, 9 lipids, 10 neurotransmitters, 11 antioxidants, 12 by-product of purine metabolism, 13 cancer biomarkers, 14 toxins in food or drinking water, 15 all of major importance for human health. In the last two years numerous papers have reviewed the graphene based sensors and biosensors from different points of view. [16][17][18][19][20][21] To mention a few, Sun et al. 22 reviewed the latest graphene-based sensors for human health monitoring. In other review, the authors discuss about the analytical performances of sensors and biosensors based on graphene-related materials in the field of clinical, environmental and food sciences. 23 An overview of recent electrochemical sensors and biosensors based on graphene and graphene-like materials for biomarkers detection was also reported. 24 An important role in graphene and graphene-based materials sensors and biosensors is played by the used electrodes. Presently, there are various strategies regarding the production and modification of electrodes (Fig. 1). 25 Cinti and Arduini 26 presented in their review different approaches for the preparation and applications of graphene-based screen-printed (bio)sensors. They also included several methods for the modification of screen-printed electrodes, most of them being appropriate to modify other electrodes as well.
As depicted above, an extensive collection of reviews about graphene and graphene-based materials in electrochemical sensing application already exists. Nevertheless, the major interest in this field justifies the present review. We start with a summarily description of graphene synthesis methods. Next, we will discuss on the latest graphene-based materials analytical performances as sensors and biosensors toward glucose, cholesterol, dopamine, ascorbic acid, uric acid, bisphenol A, cancer biomarkers and heavy metal ions covering the period of 2017-2019. For each sensor we present the limit of detection, sensitivity and stability offering a comparison between the modified electrodes. In the end, the results of the review will be concluded.

Graphene Synthesis
To this day, graphene is still an expensive material, in the "experimental" stage (single layer graphene is not yet mass-scale produced) and pretty difficult to handle but is an attractive substrate for use in sensors and biosensors due to its excellent mechanical properties, large surface area and excellent electrochemical activity having the potential to yield high-sensitivity devices. There are various strategies for the synthesis of graphene with different characteristics.  Until now, numerous papers and review articles about the synthesis methods and characterization of graphene have been reported. [27][28][29][30][31] The graphene synthesis can be realized by two main approaches: top-down (destruction) and bottom-up (construction) methods as illustrated in Fig. 2. 32 By the top-down methods (mechanical and electrochemical exfoliation, arc discharge, oxidative exfoliation-reduction, liquid-phase exfoliation and unzipping of carbon nanotubes) usually, the layers of larger precursors (graphite or other carbon-based materials) are delaminated into single, bi-and few-layer graphene. Among these, the graphene produced through the electrochemical exfoliation of graphite [33][34][35][36] has better crystallinity, lower oxidation degree, and fewer layers making it a very suitable material for electrodes. This method proved to be a very promising procedure for graphene synthesis and processing. The bottom-up methods (chemical vapor deposition, epitaxial growth, template route and total organic synthesis) construct graphene from smaller molecules. Although graphene produced by these methods are almost defect free with a large surface area, the disadvantages are the low yield, high production costs and elaborated experimental set-ups. For more information about graphene synthesis methods the readers are directed to the recent reviews of Adetayo and Runsewe 37 and of Mohamed et al. 38

Sensors and Biosensors Based on Graphene and Graphene-Related Materials
In the next paragraphs we present more details about the graphenebased materials analytical performances as sensors and biosensors toward glucose, cholesterol, dopamine (DA), ascorbic acid (AA), uric acid (UA), bisphenol A, cancer biomarkers and heavy metal ions. Sensitivity, limit of detection, repeatability and reproducibility were used to evaluate and validate the analytical performances of sensors and biosensors, thus being confirmed the quality of the results.

Glucose and cholesterol electrochemical sensors and
biosensors.-The detection and control of glucose and cholesterol are very important in clinical diagnosis due to the fact that variations in their levels can contribute to some severe diseases (diabetes and cardiovascular diseases 39 ). Biosensors go further than just diagnostics as they are providing real-time data preparing both doctors and patients for possible health issue in the immediate future. [40][41][42] Commonly, the electrochemical detection of glucose and cholesterol is realized by the enzymatic reaction between the analyte and oxidase enzyme. The non-enzymatic detection of glucose and cholesterol is usually based on the direct electrocatalytic oxidation of the analyte on the surface of a modified electrode. A stable, selective, and sensitive electrochemical sensor for glucose and cholesterol detection is still needed. The detection of glucose and cholesterol may be performed simultaneous or individual.
Ounnunkad et al. 43 developed a sensitive amperometric biosensor for glucose and cholesterol detection using a platinum/reduced graphene oxide/poly(3-aminobenzoic acid) nanocomposite film on a screen-printed carbon electrode. The biosensor presented excellent sensing capabilities in the detection of both metabolites, with wide linear range, high sensitivity, and low detection limits being suitable for real sample analysis in clinical diagnosis.
A stochastic sensors used for the molecular enantiorecognition and quantitative determination of the enantiomers of glucose in urine and whole blood samples showed good selectivity and enantioselectivity, stability in time, and high reproducibility within concentration ranges which covered the specific range of D-glucose for healthy and diabetic patients. 44 In Tables I and II we present the analytical parameters of sensors and biosensors prepared with graphene for glucose and cholesterol detection, respectively.
In addition to real-time response, stability, sensitivity and selectivity the sensors miniaturization and low-cost production needs to be resolved in order for the cholesterol (bio)sensors to be used in practical applications. Gradual fouling of the sensing area is another problem that still needs to be overcome.
Dopamine, ascorbic acid, uric acid.-Dopamine, one of the crucial catecholamine neurotransmitters, ascorbic acid, a well-known antioxidant, and uric acid, the primary end-product of purine metabolism, are compounds of major biomedical concern, playing significant roles in human metabolism. Their abnormalities in human body may indicate several serious diseases (Parkinson and schizophrenia, 61 gout 62 ) therefore their quantitative determination by electrochemistry could give proper evidences for diseases diagnosis and treatment. Several modified electrodes were successfully used in the separate or simultaneous detection of DA, AA and UA. 63 Graphene-modified electrodes were also applied conveniently to detect these biologically important compounds. [64][65][66] For an easy overview on the existing sensors based on graphene for DA, AA and UA detection we present few of the existing data in the following table (Table III).
The presented results proved that graphene-based materials could perform with good accuracy for the individual or simultaneous detection of DA, AA, and UA with great potential for future diagnosis. However, the real challenge still remains, that being the development of an economical and practical sensor which also has to be highly sensitive, selective and reliable.
Bisphenol A electrochemical detection.-Bisphenol A (BPA, 4,4'-(propane-2,2-diyl)diphenol), extensively used for polycarbonate plastics, phenol and epoxy resins manufacturing, is an endocrine disruptor which can mimic estrogen and contribute to adverse health effects on animals and human beings. 81 Due to its negative effects on human health and on the environment it is crucial to develop a rapid, simple, sensitive and precise sensor for BPA determination. Jalalvand et al. 82 presented an extensive review about the electrochemical sensors and biosensors for BPA determination in real samples. Also, they discussed about the detection of DNA damage induced by BPA.
With the advantage of low cost, simplicity and good sensibility electrochemical sensors have been widely used for BPA detection. In order to improve the sensors performance special efforts are made to find the most suited electrode modification materials. Due to their unique properties such as: good electrical conductivity, large specific area and great catalytic activity, noble metal nanoparticles are extensively used in electroanalysis. After combination with graphene the obtained nanocomposites exhibit excellent synergic effect toward BPA detection. Thus, electrodes modified with Au-Cu bimetallic nanoclusters/graphene nanoribbons, 83 AuPd nanoparticles-loaded graphene nanosheets, 84 Au nanoparticles loaded on reduced graphene oxide -multiwall carbon nanotubes 85 could detect low BPA concentrations (e.g. 8 nM 84 ) within a broad linear range (0.01−10 μmol L −1 ).
Zhang et al. 86 reported a novel electrochemical sensor for BPA detection based on reduced graphene oxide-silver/poly-L-lysine nanocomposites modified glassy carbon electrode. The high electrocatalytic activity to the BPA oxidation was determined using differential pulse voltammetry. The developed modified electrode was tested for BPA detection in drinking water with acceptable results. A facile hydrothermal method was used to synthesize a new nanocomposite material formed by molybdenum copper selenide nanoparticles decorated with reduced graphene oxide and used for the first time as a sensing material for BPA detection. 87 The sensor excellent electrochemical response opens new possibilities for the elaboration of electrochemical sensors with hybrid materials. A composite formed of graphene nanoplatelets and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate was utilized as a modifier for glassy carbon electrodes with exceptional results (detection and quantification limits of 6.4 nM and 0.02 μM; linear range 0.02-5.0 μM). 88 The electrochemical sensor proved to be stable, showing good repeatability, reproducibility, high accuracy and adequate selectivity and good response in water samples. h SPE/rGO/Ch/Fe(C 5 H 5 ) 2 /PtNPs -screen-printed electrode/reduction graphene oxide-chitosan-ferrocene/platinum nanoparticles. i AuNPs/rGO/polyamide-amine-Fe(C 5 H 5 ) 2 -Au nanoparticles and reduced graphene-polyamide-amine-ferrocene. j Silylated GO -Silylated Graphene oxide. k AuNPs/SPE/GO/AuNPs/co-mediated Ag -Au nanoparticles/screen-printed electrodes/graphene oxide (GO) and gold nanoparticles (Au NPs) co-mediated enzymatic silver deposition. l GCE/PPy/green rGO/ChOx -glassy carbon electrode/polypyrrole, green reduced graphene oxide and cholesterol oxidase. m ChOx/AgNPs/GO -Cholesterol oxidase over silver nanoparticles coated graphene oxide nanosheets. n GO-MIP -modified graphene oxide based molecular imprinted polymer.  Another study 89 reported a new, fast and highly sensitive BPA sensor obtained by a simple one step electrochemical reduction of graphene oxide and Cu 2 O. The modified electrode exhibited a low detection limit (5.3 × 10 −8 M (S/N = 3)) in the detection range from 1 × 10 −7 to 8 × 10 −5 M and was used with success for BPA detection in real water samples.
Karabiberoglu 90 developed a very sensitive and selective sensor for BPA determination using metallic copper-copper oxide and zinc oxide decorated graphene oxide (GO) to modify a glassy carbon electrode. The cyclic voltammograms were recorded on bare glassy carbon electrode (Fig. 3a); GO modified glassy carbon electrode (Fig. 3b) and Cu-Zn/GO/GC electrode (Fig. 3c) a decreasing of the oxidation peak current of BPA being observed in all cases after several cycles.
The proposed electrochemical sensor has two linear ranges within the concentrations 3.0 -0.1 μmol·L −1 and 0.35 -20.0 μmol·L −1 , being used with satisfactory results to detect BPA in baby feeding bottle, pacifier, water bottle and food storage containers.
Porous graphene functionalized black phosphorus composite was successfully used to prepare a new electrochemical sensor for BPA detection. 91 Under optimized conditions, the modified electrode showed a good linear relationship in the range of 4.3 × 10 −8 -5.5 × 10 −5 mol/L with a detection limit of 7.8 × 10 −9 mol/L (S/N = 3). A good alternative for the classical methods of analysis is represented by stochastic sensors. Bisphenols-A (BPA), -F (BPF), and -Z (BPZ) were detected in waste water samples by using three stochastic sensors based on modified nanocarbon paste, nanographene paste, and reduced graphene oxide paste with 2,2-diphenyl-1-picrylhydrazyl (DPPH). 92 Despite the good results presented above, a faster, simpler and more sensitive electrochemical method for BPA detection is still needed to be developed.
Cancer biomarkers.-According to World Health Organization, cancer is the second largest disease globally with a growing mortality rate over the past few years. For a successful treatment early diagnosis is crucial. Biosensors have a very important part in the cancer biomarkers detection being easy to use, convenient and can perform really fast analysis. In many research papers and reviews are presented different types of biosensors designed to detect cancer. [93][94][95] Here, we present the recent advances in cancer biomarkers detection using sensors and biosensors with graphene-based materials. Nanobiosensors are convenient because of their high selectivity, sensitivity, and rapid response which allow the detection of various cancer biomarkers. [96][97][98][99] An effective approach for cancer biomarkers detection is based on using metal nanoparticles-graphene modified electrodes. Many studies have evaluated the graphene/AuNPs electrochemical response for various cancer biomarkers such as three-dimensional graphene/gold nanoparticles composite for the sensitive and selective detection of tumor marker cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) 100 cobalt sulfides/graphene and gold nanoparticles served to modify a graphite screen-printed electrode and to detect the protein biomarker carbohydrate antigen 15-3 (CA15-3), 101 graphene oxide, gold nanoparticles, and specific antibodies as a diagnostic tool for the detection of tumor markers, prostate cancer, and clinical analyses, 102 graphene oxide decorated with Au nanoparticles, capable to detect androgen released by prostate cells 103 (Fig. 4). The electrochemical biosensor exhibited a detection limit of 0.5 ng/mL and a linear range of 0-110 ng/mL.
When superior performance characteristics are combined with ease of fabrication, long-term stability, good reproducibility, rapid response, and excellent specificity to cancer biomarkers, the modified electrodes are potential candidates for early diagnosis of cancer.
Electrochemical sensors for heavy metal ions.-Toxic heavy metal ions are known to be damaging to human health and when their contents surpass the natural background values in soil or water environment generate ecological and environmental degradation. For this reason, their detection by a simple, fast and sensitive method is essential. In comparison with the conventional detection methods, electrochemical sensors based on graphene and graphene-based materials have the advantages of high sensitivity and selectivity, rapid detection, and high stability being useful for heavy metal ions detection. There are several review papers referring to the recent progress in the application of graphene-based nanomaterials in the detection of some important heavy metal ions. 104−106 For example, Qiu et al. 107 have reviewed the existent literature on the optical sensing of Cu 2+ ; Pb 2+ ; Hg 2+ ; Cd 2+ ; Ag 2+ , using graphene-based nanomaterials. The performance of sensors for metal ion detection was effectively improved when graphene-based nanomaterials were employed. Based on the DTF calculations with which Yakimova et al. 108 investigated the interaction between neutral and charged heavy metal atoms (Cd, Pb, Hg) and different zigzag and armchair-edged graphene quantum dots, it was suggested a new route toward the progress of graphenebased sensing platform for the detection of heavy metal ions. They demonstrated that the most convenient adsorption site for Cd and Hg is the high-symmetry hollow site, while Pb links preferably on the bridge site.
Among the heavy metals, Cd 2+ and Pb 2+ presents severe risks to human health such as reproductive toxicity, damage to the human immune system, respiratory disorders, and negative effects on metabolism. In recent years, different modified electrodes employed for their separate or simultaneous detection have been reported. Luo et al. 109 developed a sensitive and selective "turn-on" fluorescence sensor for Pb 2+ detection using graphene quantum dots and gold nanoparticles. A new nanocomposite based on nitrogen-doped graphene and chitosan was developed and employed to modify a glassy carbon electrode used for determination of Pb 2+ cations from aqueous solutions. 110 Thinakaran et al. 111 developed a modified glassy carbon electrode employing reduced graphene oxide, carboxymethyl cellulose and glutathione for the electrochemical detection of Cd 2+ . The linear range was 2-20 nM, with a detection limit of 0.05 nM and a sensitivity of 4.5 μA/nM. Their studies indicate that the new nanocomposite represents a favorable material for the development of an inexpensive and efficient sensor for Cd 2+ detection. A porous graphene oxidepolypyrrole polymer nanocomposite modified sensor for electrochemical trace analysis of Cd (II) 112 was also developed.
The simultaneous detection of Cd 2+ and Pb 2+ was realized with several electrochemical sensors based on nitrogen-doped quantum dots graphene oxide hybrid; 113 multilayer graphene paste electrode (mixture of graphene flakes and mineral oil) modified with activated carbon; 114 graphene nanosheets with hierarchical MgFelayered double hydroxide (LDH) on the surface; 115 graphene oxide, κ-carrageenan and L-cysteine; 116 a composite film of reduced graphene oxide/carboxylation multi-walled carbon nanotubes/gold nanoparticle hybrid material; 117 a flexible Au substrate with micro-patterned reduced graphene oxide and a carbon nanotube composite. 118 The use of disposable 3D printed microfluidic chip coupled with a sensor based on epitaxial graphene on SiC for Pd 2+ detection 119 favored the reduction of reactants consumption and showed good stability and reproducibility over time proving the high potential of epitaxial graphene to be used as a sensitive sensor for heavy metals ion detection.
MnO 2 is an environment-friendly, low-cost material, which has been widely studied in the sensors area. Using a hydrothermal method, Wu et al. 120 synthesized a novel nanocomposite material (nitrogen doped reduced graphene oxide (p-phenylenediamine as nitrogen source) and MnO 2 ) which was employed to modify a glassy carbon electrode following applied for Hg 2+ detection with low detection limit (0.0414 nM) and good sensitivity (72.16 mA/mM). The modified electrode also denoted high stability and reproducibility.
A glassy carbon electrode modified with electrochemically reduced graphene oxide was used as a sensitive sensor for the rapid and simultaneous detection of Pb 2+ and Hg 2+ . 121 The limit of detection of the modified electrode toward Pb 2+ and Hg 2+ was 0.2 ng/mL and 1 ng/mL respectively, in the linear range of 1∼1000 ng/mL(for Pb 2+ ) and 1∼1000 ng/mL (for Hg 2+ ). The proposed modified electrode may be used for qualitative and quantitative detection of heavy metal ions in various real samples.
The electrodes presented above have significant merits including high stability and reproducibility however, their sometimes inconvenient fabrication and high production costs may limit their applications to some extent.

Conclusions and Outlook
To sum-up, graphene and graphene-based nanomaterials are excellent electrode materials, their characteristics making them extremely competitive in comparison with the classical electrodes, and there are still plenty of opportunities for scientific research and applications to be developed using graphene based materials and devices. The exceptional properties of graphene-based materials such as high electrochemical activity, ease of surface functionalization, and excellent electron transfer properties have enabled them to be used with excellent results to detect the analytes with improved sensitivity and selectivity. We reviewed the application of graphene and graphene related materials as sensors and biosensors for the detection of glucose, cholesterol, dopamine, ascorbic acid, uric acid, bisphenol A, cancer biomarkers and heavy metal ions. Most of the presented results proved to be functional to determine the specific analytes in complex real samples.