Advanced Graphene-Based Materials for Electrochemical Biomarkers and Protein Detection

Abstract

In this chapter, recent advances in the field of graphene materials-based (bio)sensors that are used for biomarker and protein electrochemical detection are presented. Approaches related to the synthesis of electrode material for (bio)sensors construction as well as to their morphological and structural characterization, are highlighted, pointing out the advantages of using graphene-based materials for (bio)sensors applications. This chapter focuses on obtaining graphene-based electrodes, detecting biomarkers and proteins, and evaluating the performance of the sensors. Different methods for designing sensors for a large variety of biomolecules are described and comparatively discussed. In order to increase their electroanalytical performances, such as sensitivity, selectivity, detection limit, linear range, and stability, the research performed in the last years was focused on different types of graphene structures including graphene oxide, reduced graphene oxide, graphene nanofoams, graphene nanocomposites, different functionalized graphene, etc. The detection of analytes including neurotransmitters and neurochemicals (dopamine, ascorbic acid, uric acid, serotonin, epinephrine, etc.), hydrogen peroxide, and proteins, have been discussed. The studies related to electrochemical (bio)sensors are presented in three subchapters, and the key results—electroanalytical performances—of the sensors are summarized. The final chapter provides the conclusions derived from the comparative analyses of different approaches related to these types of (bio)sensors based on graphene materials.

1. Introduction

In the last decade, the necessity for rapid, simple hand-held testing devices in medicine has prompted the development of biosensors for clinical purposes. Biological sensors were developed as optical, electrical, piezoelectrical devices or systems, which consist of biological and electronic components and are able to discover and detect biological compounds like nucleic acids, proteins, enzymes, and genes. Nowadays, biosensors are known as devices that are employed for qualitative and quantitative detection of biological analytes [1]. The biomolecules, the biological structures, or the microorganisms can play the role of biological analytes. As it is well-known, an electrochemical (bio)sensor involves three components, which are responsible for (i) the recognition of the analyte and the signal formation, (ii) the transducer of the received signal, and (iii) the reader device [2]. Owing to an interdisciplinary combination of approaches from physics, chemistry, biology, nanotechnology, and medical science, the achievements in the biosensor field are impressive. Consequently, they are becoming essential devices developed for diagnosis of life-threatening syndromes [3, 4].

Electrochemical sensors are of high interests in different applications because of their great sensitivity, selectivity, inexpensive and simplistic production, and facile miniaturization [5, 6]. They depend on the use of sensitive biological molecules immobilized on the surface of solid electrodes that are able to capture target molecules by specific recognition [2, 3]. This process, at the electrode surface, occurs with a reaction signal. The modified electrode transforms the produced chemical signal into a measurable electrical signal, such as current, voltage, conductivity, impedance, etc. This way, the technique enables both the qualitative and quantitative analysis of target species [7, 8].

Electrochemical biosensors have some advantages as compared to other biosensor categories. First of all, the theory behind it is well-developed, and it involves a facile design based on simple structures for easy measurements [5, 9]. As compared to other analytical methods including fluorescence [10, 11] colorimetric [12], and chemiluminescence methods [13, 14], electrochemical biosensors can be used in many important fields such as biomedicine, pharmaceutical industry, food industry, and environmental analysis [3, 4, 15]. In order to increase the sensor performances with respect to detection limits, sensitivity, selectivity, time stability, and linear detection range and to lessen the time of response, recent research was focused on developing a new preparation method for biosensor electrodes using different electrode materials.

Thus, due to their low cost, compared to other materials such as Pt and Au metals, and because of their good conductivity, a variety of modified electrodes incorporating carbonaceous materials [16], such as graphite [17, 18], carbon aerogel [19, 20, 21, 22], glassy carbon [23, 24, 25, 26, 27, 28], graphene [25, 29, 30, 31, 32, 33], carbon fiber [34], and screen-printed electrode [35], have been developed for sensing of numerous biological compounds. Also, various materials including metals nanoparticles Au [25, 36, 37, 38, 39], Ag [40]; phosphates such as zirconium phosphates [28, 41] and titanium phosphates [27]; mediators [42], polymers (PANI [37, 43], chitosan [44, 45, 46]), enzymes (HRP) [17, 23, 47], plasmodium falciparum lactate dehydrogenase (PfLDH) [48], cellobiose dehydrogenase (CDH) [20]; oxides (α-Fe₂O₃ [49], Al₂O₃ [50], ZnO [36, 51], Cu₂O [52]), and non-metals (N [53]) have been used widely to modify different carbonaceous electrode materials.

Among the carbonaceous modified electrodes, those based on graphene matrix, i.e., graphene (G), graphene oxide (GO), and reduced graphene oxide (rGO) [54, 55], exhibit important advantages over the conventional ones. This is because the latter classes of materials, present significant drawbacks that range from limited active surface areas easy inactivation and high cost of production. G structure is composed only of carbon atoms that are all sp²-hybridized and organized in a single atomic layer. Every carbon atom belonging to G is bonded to three other neighboring carbon atoms using three of its valency electrons, and the fourth electron is delocalized, thus facilitating the electrical conductivity of G [15]. The use of G for sensors’ development is attributed to its additional unique features that include its ease of production; good chemical, morpho-structural, and mechanical characteristics, joined with other atypical properties including the high surface area to volume ratio, exceptional optical characteristics, outstanding carrier mobility, and extraordinary electrical and thermal properties as compared to those exhibited by other carbon allotropes [56]. Besides its exceptional properties, G can be functionalized easily, and many of its biomedical applications can benefit from non-toxic, biocompatible, and water-dispersible G layers obtained by chemical functionalization with different ligands [57].

The grafting of functional groups on the G surface structure is extremely reliable for binding molecules so as to further examine their communications with well-defined targets. In this respect, oxidized G presents one of the best solutions to obtain surface charges by anchoring on its surface oxygen-containing substituents like hydroxyl, carbonyl, carboxyl, and epoxide groups, which make it easier to understand the future specific interactions that take place on the surface of the G structure. Thus, G and its oxidized derivatives (GO or rGO) combine well-known properties of the carbonaceous structures, i.e., the great electric and thermal conductivities, mechanical strength, and impermeability to gases, with those related to the enhancement of their surface response after interaction with specific molecules. Accordingly, G-based materials are desired because of their essential functional groups such as those found in GO or N-G, very beneficial and effective in aligning with biomedical and related fields [58]. This is the reason why, in the last years, investigations were focused on different types of G structures including GO, rGO, G nanofoams, G nanocomposites, and different functionalized G derivatives [59], and the biosensor applications are now focused on biomarkers and protein detection.

In this chapter, the latest developments in the domain of biosensors, based on G materials, are described. Details about the synthesis and their morpho-structural investigations are also provided. Different approaches for designing sensors for a great variety of biomolecules are described and discussed. The detected analytes contain neurotransmitters and neurochemicals (dopamine, ascorbic acid, uric acid, serotonin, epinephrine, etc.), hydrogen peroxide, proteins, DNA, glucose, and others. The aim was to highlight biosensor performances for glucose, hydrogen peroxide, neurotransmitters and neurochemicals, and cancer and disease biomarkers detection. A special importance was accorded to dopamine, ascorbic acid, uric acid, serotonin, and epinephrine detection. The conclusions resulting from the presented research on the biosensors are summarized in the last subchapter.

2. Synthesis, structure, and morphology of graphene materials for sensing applications

G materials have been widely promoted due to their suitable properties and versatility in biomedical applications as like biosensors, diagnosis-imaging, and drug delivery [58]. G consists of a one-atom-thick carbon structure that forms a 2D hexagonal honeycomb nanomaterial (Figure 1a), but besides the pristine G materials, the functionalized G structures (containing hydroxyl, carbonyl, carboxyl, and epoxide groups on the edge or plane of the nanosheets) such as GO (Figure 1b) and rGO (Figure 1c) are tremendously involved and tested in the same biomedical application fields. As a convention, the limits of 2D G are accepted to have the thickness of up to 10 atomic layers (i.e., a few nanometers) and the lateral size larger than 10 nm up to more than 20 μm [61]. Those having less than 10 atomic layers and a few thousand nanometers in size will be termed as nanosheets. The properties that make G materials suitable for various purposes can be adjusted by selecting the appropriate synthesis pathway. If the target is represented by the sensing applications, then the obtained structures are desired to recognize entities having similar physical properties and to provide information about them [58].

Figure 1.

Suggested structural models for monoatomic layered nanosheet of G (a), GO (b), and rGO (c); the blue square (b) depicted the electrical conductor oasis of GO. The red ovoid (c) depicted electrical insulator oasis of rGO. Reproduced from Ref. [60] with permission from the Royal Society of Chemistry.

One very important parameter of G for electrochemical sensing applications is its semiconductor behavior with zero band gap energy, which shows that G has electrical conductivity much greater than that of copper, resulting from its extended π-π conjugation of sp² hybridized carbon atoms. In the same way, rGO is considered an electrical conductor that contains electrically insulating oases (Figure 1c), whereas GO is an electrical insulator with electrically conductive oases (Figure 1b). The electrical insulation behavior of GO results from the presence of sp³ hybridized carbon atoms that are involved in anchoring oxygen-containing groups on the edges or planes of GO/rGO nanosheets. Among them, the carboxyl and epoxide groups are very commonly involved in biomolecular immobilization for biosensor construction [58].

Generally, the synthesis of G materials can be grouped into two main classes: bottom-up and top-down methods [62]. The representative pathway for the first class is carbon vapor deposition (CVD) that consists of a G growth on catalytic support (e.g., nickel or copper) using a carbon-source gas (e.g., CH₄, CH₄/H₂ mixture, C₂H₄) at a high temperature (around 1000^°C). Free-standing monoatomic layer G as a crystal sheet with the size of catalytic metallic support is released after support etching [62].

Among the top-down methods is the one proposed by Paton et al. [63], which enables the obtaining of G nanosheets in a large-scale production method by physical exfoliation based on shear mixing of graphite. Even though, the obtained G has a high yield, good conductivity, and strong mechanical properties, its poor dispersion in common solvents (e.g., water and ethanol) is the primary disadvantage [64]. The most efficient ways to overcome this drawback is to use a dispersing agent (e.g., amphiphilic polymers, alkylamines, and molecules with hydrophilic carboxyl groups) or to generate organic functional groups (e.g., hydroxyl, carbonyl, carboxyl, and epoxide) on the planes and the edges of G sheets. The most known of such methods are Hummer [65] and its derivates [66], which are based on chemical oxidative exfoliations of graphite using concentrated sulfuric acid (H₂SO₄) and sodium nitrate (NaNO₃) as the reaction medium combined with potassium permanganate (KMnO₄) as oxidant. But toxic and hazardous gases (e.g., NO₂ and N₂O₄) are generated if the reaction temperature overcomes certain limits. Marcano et al. [67] proposed an improved method of Hummers that consists in an oxidative exfoliation, in which phosphoric acid (H₃PO₄) was involved. Better efficiency in GO production and no hazardous gas generation were claimed. By using sonication and a lower reaction temperature, a more controlled sono-oxidative-exfoliation process is obtained [60, 68]. A new GO preparation route is proposed by Peng et al. [69], who used the potassium ferrate (K₂FeO₄) as the oxidant and concentrated H₂SO₄, as acidic medium for the reaction. Besides the higher quantity of GO that can be obtained in comparison with the other reported pathways, the approach presented a simple way of preparation with the additional benefit brought about by the recycling of H₂SO₄. In another pathway version promoted by Yu et al. [70], an oxidant mixture of K₂FeO₄ and KMnO₄ was employed with boric acid (H₃BO₃) as a stabilizer in H₂SO₄. By involving such liquid phase exfoliation methods, GO nanosheets that are proper to form stable aqueous suspensions can be obtained. For sensor construction, the obtained 2D GO nanosheets have to be capable to form stable individual 3D structures as nanobricks or to form (nano)composites/modified structures by combination with other components.

Generally, the 3D structures of G materials can be grouped into three main classes: films (i.e., supported membranes), membranes (i.e., unsupported films), and porous structures (e.g., foams, aerogels/xerogels, frameworks, etc.).

In the case of G obtained by using the CVD technique and flat metallic support, a large-area continuous 2D film or membrane can be obtained after etching the support by using acid solutions. But, if the template is a porous 3D metallic structure (e.g., nickel foam, copper powder, magnesium, or aluminum oxide nanoparticles), a 3D G, as a coating film of metallic framework support, stabile hollow or macroporous scaffolds without metallic part can be achieved after the metallic support is etched [71]. Due to the lack of promoter-link groups on the surface of G it is difficult to obtain stable 3D structures if non-functionalized G nanosheet/nanoflake suspensions are involved. Besides, although the individual G nanosheets exhibit a very high electrical conductivity, because of the poor connection between the resulted 3D materials, a lower conductivity is measured. To overcome this drawback, the G nanosheets should be functionalized. The most known and used functionalized G material is GO. In this case, the most common methods for obtaining films are vacuum filtration, dip or drop-casting, spraying, screen printing, and the Langmuir–Blodgett method [62]. Due to the strong connection between functional groups of neighboring GO nanosheets, such as hydroxyl, carboxyl, carbonyl, and epoxy (see Figure 1b), very stable 3D laminar structures are formed as 3D GO films on the support materials after their drying. By removing (generally by a simple exfoliation) the forming support, stable free-standing GO membranes (i.e., unsupported films) can be obtained [68].

Besides this, Cotet et al. [60] proposed an elegant method that enables the harvesting of a stable membrane formed as a self-assembled pellicle at air-liquid interface after performing an isopycnic separation of a certain GO fraction. A proposed schematic distribution of functional groups present in the obtained dried GO membrane is illustrated in Figure 2.

Figure 2.

Suggested 3D laminar GO structure with intraplanar (ovoid) or interplanar (rhombic) interactions, hydrogen bonding involving H₂O (square) or not (rectangular), and ester linking (circular). Reproduced from Ref. [60] with permission from the Royal Society of Chemistry.

For a deep understanding of the interaction processes between GO nanosheets that build up such a GO membrane, an assessment of the zeta potential was performed for initial GO suspension [72]. A variation in time of the zeta-potential values with high negative values (i.e., −63 and −67 mV) at about 3 weeks from the GO synthesis, followed by a stabilization (at about −45 mV) after 4–6 weeks, was observed. These high values of negative charge indicated a high density of functional groups present both on the plans and edges of GO nanosheets [72]. The presence of high number of functional groups is beneficial for functionalization due to the potential of attaching specific groups or structures including sensitive biological molecules that can be involved in targeted sensing activity.

Porous 3D structures from the class of aerogel/xerogel/cryogel are obtained by drying in supercritical conditions of liquid CO₂, in ambient conditions or by freezing-sublimation of wet hydrogels. A simple way to obtain hydrogels with GO nanosheets in their structure was promoted by Worsley et al. [73]. A GO suspension was involved as reaction medium for the polycondensation reaction of resorcinol with formaldehyde catalyzed by sodium carbonate (Na₂CO₃). In another approach based on a hydrothermal route [71], the added carbohydrates (e.g., glucose, cyclodextrin, and chitosan) played both the role of morphology-oriented agents and of reduction agents, which define the structural, physical, and electrochemical properties of the obtained 3D interconnected-pore aerogel.

A cheaper method for 3D GO porous structure synthesis without the necessity of complex equipment and supporting substrates is the room-temperature vacuum centrifugal evaporation of a properly concentrated GO suspension, which is described in the literature [71].

During the research and testing of GO materials for sensing applications, a very important step is represented by the reduction process that partially restores the structure and electrical properties of G. In this way, a conversion from insulating GO into electrically conductive rGO occurred with the increase of the C:O molar ratio. These reduction processes can be thermal, chemical, electrochemical, irradiation-induced, etc. [62, 68]. Among these approaches, the thermal and chemical reduction methods are identified as the most used techniques, and we will therefore refer to these methods later in this chapter.

In the case of GO materials modified with bio-structures, particular sensitivity (e.g., pH and temperature) of this kind of class of substances have to be taken into account. Moreover, if the modification will be carried out with more sensitive bio-structures, the reduction process, which is harsh, should be performed prior to this process. Additionally, it is known that the higher electrochemical properties of G materials are due to their intrinsic favorable properties represented by the π-π stacking interactions. These can enhance electrochemical signals and the irreversible anchoring of catalytic sites onto G materials. Thus, it is important that the new 3D structures formed present these structural advantages [68]. A method used to improve these properties is the doping of G material with nitrogen. Shao et al. [74] obtained nitrogen-doped G by exposing G materials to nitrogen plasma. The as-obtained N-doped G materials produce the best result in the detection of hydrogen peroxide (H₂O₂) due to nitrogen and oxygen-containing groups. Interesting results were obtained by Fort et al. [21], who observed that the presence of Fe in the carbon aerogel matrix led to the formation of G-like structure as single-layer nanosheets (Figure 3), which increased the conductivity of the electrode materials and the electroanalytical performance of the obtained modified electrode for H₂O₂ detection [21].

Figure 3.

TEM (transmission electron microscopy) images of Fe-doped carbon aerogels showing G-like nanosheets (a and b); carbon nanoribbons, which are isolated (c) or around the Fe nanoparticles (a and c); and carbon nanotubes (d). Reproduced from Ref. [21] with permission from the Elsevier.

The graphitic nanostructure formation in the presence of Fe and Bi was observed by Rusu et al. [75]. The graphitic structure obtained (Figure 4) is relevant in charge transport and thermo-chemical processes.

Figure 4.

Graphitic nanostructures evidenced in carbon matrix that contains bismuth-iron nanoparticles: (a) TEM and HR-TEM (high resolution-Transmission Electron microscopy) images after heating at 900°C with two highlighted regions during the in situ TEM experiment, (b) electron tomography results on a grain representative for the graphitized sample. On the left, a 2D projection extracted from the tilt series was used to reconstruct the grain volume; on the middle and right, two orthoslices took at different depths within the reconstructed volume evidencing a tubular morphology of the graphitic structure having a length of roughly 120 nm [75]. Reproduced from Ref. [75] with permission from Cambridge University Press.

del Pino et al. showed that the use of the ultraviolet laser irradiation of flexible free-standing GO membranes in the ammonia-rich atmosphere and liquid environments generates significant integration of nitrogen groups, especially amines, in a partially reduced GO structure [68]. This method allowed for the obtaining of films with controlled geometry of reduced areas up to hundreds of square centimeters of N-doped rGO materials with high potential for (bio)sensing applications. So, flexible electrodes prepared on flexible polymeric supports with 10 μm thickness and low resistivity (6 × 10⁻⁴ Ω m) were obtained by an innovative laser reduction protocol [76].

There are various chemical agents that are used in GO reduction: hydrazine (N₂H₄), alcohols, sodium borohydride (NaBH₄), hydriodic acid in acetic acid (HI/CH₃COH), sodium/potassium hydroxide (NaOH, KOH), iron/aluminum powder (Fe/Al), ammonia (NH₃), hexylamine (CH₃-(CH₂)₅-NH₂), sulfur-containing compounds, hydroxylamine hydrochloride (HONH₂·HCl), urea (CH₄N₂O), manganese(II) oxide (MnO), enzymes, vitamin C, bacteria, etc. [77]. However, the most used reducing agent is hydrazine, which allows for the obtaining of rGO materials with a C:O of about 12.5:1 and a conductivity of 99.6 S cm⁻¹ [78]. But, because of its toxicity, green alternatives are encouraged to be developed. From these, reduction with alcohols [79], hydriodic acid [80], and vitamin C [81] are reported [82].

Thermal reduction of GO materials consists of organic (i.e., hydroxyl, epoxy, carbonyl, carboxyl, etc.) group removal as O₂, CO, CO₂, and H₂O by the aid of high temperatures (>120^°C) [83]. Because hazardous reagents are not involved, this reduction method is considered to be safer than certain chemical reduction approaches. The degree of reduction can be adjusted by controlling the reaction temperature and the process time. A high temperature (of about 400–1050^°C) and an oven equipped with an inert atmosphere (i.e., Ar or N₂) or in hydrogen flow are required to obtain rGO materials with high electrical conductivity [62]. Nonetheless, even by the use of these methods, the materials obtained show a low surface and a smaller number of insulated oases (Figure 1c) due to the remaining functional groups from rGO materials that could be further involved in applications related to the sensing electrode construction.

To the best of our knowledge there has not yet been established a direct relationship between the morphology-structure of a composite and its sensing properties. However, there are several studies reported in which particular correlations between the structure and sensing properties were made by keeping in mind the possibility of improving the sensing performances as a result of understanding the structural particularities.

Besides the excellent electrical conductivity, another remarkable property of G, when used in sensing applications, consists of its high specific surface area (theoretical specific surface area ~2630 m²/g), which promotes the attachment of (bio)molecules, polymers, or nanoparticles to the G surface by exposing all carbon atoms to sense biomolecules. These results indicate an increased G’s sensitivity as biosensor [84]. After synthesizing cobalt oxide (Co₃O₄) nanowires using a hydrothermal method based on 3D G foam grown by CVD, Dong et al. used this composite (G/Co₃O₄) as a monolithic free-standing electrode for free-enzyme electrochemical detection of glucose. The porous structure of the G foam proved to play the role of a scaffold for the sensors fabrication [85]. A double-layered membrane of rGO-and-polyaniline (PANI) nanocomposites (to enhance the sensitivity of sensor) and molecularly imprinted polymers decorated with gold nanoparticles was prepared by Xue’s research team in order to obtain an electrochemical serotonin sensing interface. When the morphology of the material was studied by SEM (Scanning Electron Microscopy), a uniform distribution of rGO/PANI nanocomposites spheres with the diameter of the single sphere of approximately 93 nm was observed. A rough and foliated structure embedded with AuNPs was observed. This indicates the achievement of imprinted membrane embedded with AuNPs, which led to an increased conductivity and electrocatalytical activity of the membrane [37]. By employing electrophoretic deposition in a magnesium salt/GO electrolyte, Akhavan et al. prepared GO nanowalls with extremely sharp edges and preferred vertical orientation, and then they deposited them on a graphite electrode. The results revealed a uniformly deposited film of GO nanowalls on the electrode surface. Interesting petal-like G nanosheets with lateral sizes of ∼500 nm and very sharp edges (1–15 nm thickness) were observed. Because of the formation of a large fraction of graphitic edge-plane defects that can lead to the obtaining of a higher surface activity than the G nanosheets placed parallel to the substrate, the authors speculate that such vertical nanosheets may exhibit distinctive electrochemical properties [86]. By SEM investigation [60] of free-standing self-assembled GO membranes (Figure 5), a continuous wavy feature (Figure 5a) and a compact layer-by-layer stacking (Figure 5b) were observed at the surface and the cross-section of obtained dried GO membranes.

Figure 5.

SEM images of the self-assembled GO membrane surface (a), and its 3D view (see the inset), the cross-section (b) obtained by dispersing a certain GO fraction on an open area framework support and harvesting it after 15 min, and the average membrane thickness evolution against the self-assembly time (c) [60]. Reproduced from Ref. [60] with permission from the Royal Society of Chemistry.

In the meantime, in the case of this preparation pathway, by changing the time used for self-assembled process from 15 to 120 min, GO membranes with about 2.85 μm and 11.40 μm thickness, respectively, were obtained. This kind of GO membrane type could be properly reduced (e.g., by protected laser irradiation, Figure 5, [76]) to obtain 3D rGO membranes or films with controlled geometry, reduction degree, and morpho-structural properties suited for biosensor construction.

3. Biosensor applications

As already reported, the attractive properties of G-based nanomaterials can be successfully used in biomedical applications as electrochemical-based biosensors with improved analytical performance with respect to low detection limits, selectivity, high sensitivity, low response time, reproducibility, and large linear detection range [54, 56, 59, 71]. In addition, a significant improvement in the sensors is mechanical properties, relating to flexibility. An important work focused on the importance of flexible sensors was reported by Giaretta et al. [87]. They compared the biosensors performances based on the obtained material substrates. It was concluded that the polymeric substrates are the cheapest, but their performances were inferior when compared to those of the other substrates obtained from carbonaceous materials. This is due to the poorer electrical conductivity, reduced permeability and lack of porosity, respectively.

A bioelectrochemical sensor transforms a biological modification occurrence into an electrical signal. Hence, the working principle is based on the transfer of a negative elementary charge (the electron) between the G interfaces and species that present electrical activity. The species can be either the molecule to be analyzed or a species whose electrochemical measured signal can be associated with the existence of the target analyte.

3.1 Biomarkers and protein detection

Real-time quantitative monitoring of biomarkers, which represent a particular class of biological compounds whose presence in serum and saliva shows a relationship with a certain disease, has become essential for early disease detection, leading to adapted treatments and investigation of treatment efficiency. Guo et al. synthesized Ni nanosheet/G composites, which have an exceptional electrocatalytic signal for the detection of L-alanine by using a direct current (DC) arc plasma jet CVD method [88].

The obtaining of 1 to 10-layered G nanosheets, G nanoribbons, and core-shell Ni/G nanosheets by the above-mentioned method was evidenced. These G-based composites were used as sensors to detect proteins. It was revealed that the higher value of the specific surface area of the G, the higher adsorption ability for L-alanine, and the good transfer of the electron between the Ni nanosheet/G composite and the surface of glassy carbon electrodes (GCE) had considerably improved the performances of the obtained sensor (Table 1). Furthermore, by means of the obtained sensor, without enzyme presence, the direct electrooxidation process of amino acids was achieved [88].

Electrodes	Analyte	Linear range	Sensitivity	Detection limit	Ref.
Ni nanosheet/G/GCE	L-alanine	1–7 μM; 0.1–1 μM	2.15 A M^−1; 8.34 A M^−1	0.1 μM	[88]
(His)-tagged (AChE)	Paraoxon	10 μM–1 mM	2.23 mA M^−1	6.5∙10^−10 M	[89]

Table 1.

Electrochemical parameters of some G-based biosensors for different analytes detection.

GCE—glassy carbon electrodes, AChE—acetylcholinesterase.

Zhang and co-workers developed functionalized graphene oxide (FGO), which exhibited high affinity to (His)-tagged acetylcholinesterase (AChE) for paraoxon manufacture, an acetylcholinesterase inhibitor, biosensors [89]. In order to estimate the functionalization of GO and their capacity to be involved as multipurpose enzyme immobilization nanomaterials for the bioelectrochemical sensor design, the AChE was nominated as being a perfect enzyme. The authors evidenced the existence of an optimum amount of Nα,Nα-bis (carboxymethyl)-L-lysine hydrate (NTA-NH₂) and that Ni²⁺ (Ni-NTA) needed to attach on the GO surface. This connection has succeeded to an increased enzyme loading, which lead to an enhanced electrocatalytic activity and sensitivity. This work proved excellent stability, for both short-term and long-term, being the effect of the stable binding between Ni-NTA and His-tagged AChE. The paraoxon concentrations also influence the inhibition response, and a low detection limit value was reached (Table 1). The authors showed that the obtained FGO composite can be changed to multipurpose biosensor construction [89].

3.1.1 Glucose

Glucose, a monosaccharide, serves as an energy source and metabolic fuel and is involved in the processes of photosynthesis and respiration in most organisms. The high blood-glucose concentration, recognized as hyperglycemia, or the reduced glucose presence, identified as hypoglycemia, is caused by the effect of insufficiency of insulin in the body, known as Diabetes mellitus, an incurable disease. By monitoring the glucose concentration in blood, as an illness marker, it is possible to extend life expectancy. Thus, people with diabetes, people who have problems with glucose concentration in blood, can manage episodes of hypo- or hyper-glycaemia, hence providing improved control over their conditions and avoiding some of the incapacitating side effects. Moreover, by monitoring the glucose, the patient’s treatment strategies can be optimized, and also, the effect of medications, physical exercise, and nutrition on the patient can be controlled.

Xue and co-workers prepared a biocompatible AuNPs/PPy/rGO nanocomposite that demonstrated exceptional electro-catalytical activity toward O₂ reduction [37]. By encapsulating glucose oxidase (GOD) via chitosan (N-deacetylated derivative of chitin) cross-linking in the obtained composite, AuNPs/PPy/rGO/GOD/chitosan modified electrode was fabricated. The developed electrochemical sensor revealed a linear range for glucose from 0.2 to 1.2 mM with a good sensitivity value of 123.8 mA M⁻¹ cm⁻² (Table 2). The obtained performances were correlated with (i) the high surface area and high electric conductivity of rGO, (ii) good shielding of PPy and fixing capacity on rGO surfaces, (iii) biocompatibility of the dispersed small AuNPs and their electron transfer promotion ability. The authors showed that the enzyme, GOD, has a good connection with AuNPs/PPy/rGO nanocomposite, which was proved by the fast electron transfer between the used enzyme and electrode. Also, between the electrode surface and the enzyme, there seems to be a rather low barrier. Furthermore, the achieved AuNPs/PPy/rGO ternary nanocomposites can be favorable for encapsulating some other biomolecules, indicating, thus, a potential substitute in constructing multipurpose bioelectrochemical sensors and additional devices [37]. Chitosan, due to its uncommon mixture of properties including the exceptional membrane-forming capability, good bond, high mechanical strength and water permeability, and extraordinary biocompatibility, is one of the best-suited biopolymers for the surface-deposition of nanocomposite for the electrode fabrication [45, 46, 92, 93].

Electrodes	Analyte	Linear range; /mM	Sensitivity	Detection limit	Ref.
AuNPs/PPy/rGO/GOD/CS	Glucose	0.2–1.2	123.8 mA M^−1 cm^−2	—	[37]
GCE/rGO/PTZ-O/GDH	Glucose	0.5–12	42 mA M^−1 cm^−2	—	[90]
rGO/PAMAM/Ag	Glucose	0.032–1.89	75.72 mA M^−1 cm^−2	4.5 μM	[40]
3D G/Co3O4	Glucose	Up to 0.08	3.39 A M^−1 cm^−2	25 nM	[85]
Pt-CuO/rGO	Glucose	Up to 12	3577 mA M^−1 cm^−2	10 nM	[35]
Cu2O/GNs	Glucose	0.3–3.3	0.28 A M^−1 cm^−2	3.3 μM	[52]
	H2O2	0.3–7.8	—	20.8 μM
Au nanocubes/G	Glucose	0.1–0.8	221.0 mA M^−1 cm^−2	—	[91]

Table 2.

Electrochemical parameters of some G-based modified electrodes for glucose and H₂O₂ detection.

GCE—glassy carbon electrodes, CS—chitosan, His—histidine, GOD—encapsulated glucose oxidase, AuNPs—gold nanoparticles, PPy—polypyrrole, GDH—glucose dehydrogenase, PTZ-O—phenothiazone, PAMAM—poly(amidoamine), GNs—G nanosheets.

With the aim of encapsulating redox enzymes, rGO film with adsorbed phenothiazine represents another composite that was synthetized by Ravenna et al. [90]. The prepared composite was very efficient for the electron transfer process between flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase and the obtained modified sensor. The study showed that for the glucose oxidation process, the determined redox potential was lower than 0 V vs. Ag/AgCl reference electrode. Furthermore, the obtained rGO-based biosensor presented an increased value of sensitivity and a large linear range for glucose detection (Table 2). Also, the obtained biosensor achieved reasonable reproducibility and stability, a great selectivity for different interfering compounds. The results demonstrated a promising sensor for several bioelectrochemical applications [90].

Luo and co-workers showed that by combining the rGO with poly(amidoamine) and silver (rGO–PAMAM–Ag), the newly developed nanocomposite offers an exceptional microenvironment to assure the direct electron transfer process of GOD enzyme fixed on the modified surface of GCE [40]. Besides, it was confirmed that the developed arrangement can preserve a great electrocatalytic activities of the enzyme. By using GO self-assembled with PAMAM-G3.5, as a growth pattern, and microwave irradiation, rGO-PAMAM-Ag new nanocomposite electrode material was prepared. Then, based on this type of sensitive nanocomposite, a biosensor for glucose detection was manufactured. A high value of sensitivity, a low value of the detection limit, and a wide linear range, were the analytical performances of the obtained biosensor (Table 2). The authors also showed that the interference between the signals originating from uric acid (UC) and ascorbic acid (AA), which are regularly detected in blood fluids together with glucose, is insignificant compared to the signal attained by the glucose biosensor. The achieved characteristics recommended the obtained rGO-PAMAM-Ag nanocomposite as an innovative exceptional electrode material for the construction of glucose biosensors, involving a direct electron transfer process [40].

An interesting study is related to the involvement of an easy synthesis path for 3D G/Co₃O₄ composite production, which was further used as a sensor for glucose [85]. Thus, the synthesis process was based on the (i) Co₃O₄ nanowires achievement by in situ hydrothermal synthesis, and (ii) CVD growth of G foam. Through this study, it was shown that Co₃O₄ nanowires possess high crystallinity and constant diameter and form a compact nanomesh covering the 3D G matrix, which can function as a free-standing electrode due to the higher mechanical strength of G. 3D G/Co₃O₄ obtained composite was employed as electrode material and the acquired results proved significant in performance as sensor for glucose and as a supercapacitor. These results were based on the synergistic incorporation of the two, G and Co₃O₄, nanomaterials and represent an important step for developing enzyme-free ultrasensitive sensors for glucose. Co₃O₄ nanowires proved excellent electrochemical and electrocatalytic properties (Table 2). The authors showed that the 3D multiplexed and extremely conductive matrix provided by the defect-free G foam depicted fast electron transfer and conduction, and offered a high available active surface area. In addition, the open pores of 3D G/Co₃O₄ composite were found to be advantageous to ion diffusion and transport kinetics. Thus, the G foams showed unique morphological properties, and these could serve as 3D supports for embedding great capacity to homogeneously bind metal oxides with well-defined properties (size, shapes, and crystallinity), as demonstrated by Dong et al. [85]. Agglomeration, which represents a regular fact of metallic oxide synthesis, is no longer a problem. The multifunctional character and the improved performance of composites based on G and metal oxides were achieved thanks to the synergies between components, representing a positive aspect for developing novel applications [85].

In their work, Dhara et al. presented an easy and low-cost fabrication process with no enzyme-limited screen-printed electrodes [35]. Thus, it was possible to synthesize Pt-CuO/rGO nanocomposite electrocatalyst by a one-step chemical reduction. The electronic microscopy measurements of the composite revealed the nanocubes structure for Pt, and nanoflowers structure for CuO. The obtained sensor for glucose oxidation process exhibited exceptional sensitivity (Table 2) and good selectivity. The acquired linear response for glucose and the detection limit value are presented in Table 2. When the newly developed sensor measured the glucose quantity in blood sample, the results could be considered as being satisfactory [35].

Another study that is worth mentioning was performed by Liu et al. and was focused on glucose detection. They demonstrated that by the use of a non-enzymatic sensor composed of G nanosheet-covered Cu₂O nanocubes (Cu₂O/GNs), good sensor’s characteristics for glucose oxidation could be obtained (Table 2) [52]. They also showed that Cu₂O/GNs hybrids can be produced by an easy technique at low temperatures, and additionally, an outstanding electrocatalytic activity for electroreduction process of H₂O₂ can be obtained (Table 2). Therefore, electroanalytical detection of glucose and H₂O₂ was achieved, and the developed sensor revealed low limits of detection, good selectivity, good linear response, and a large detection range due to the enlarged electroactive surface area and the enhanced capacity for electron transfer of the fabricated G-hybrid nanostructure. Furthermore, the authors affirmed that due to the surface covered by G nanosheets, the Cu₂O/GNs composite revealed enhanced electrochemical stability related to that acquired for Cu₂O nanocubes only. The innovative Cu₂O/GNs nanocomposite led to new possible applications in different categories of biosensors, such as sensitive electrode materials, bioelectronic devices, and (electro)catalysts [52].

By a simple electrodeposition method without any template, Chu et al. synthesized in situ an originally designed Au nanocubes/G composite film [91]. The obtained composite film possessed exceptional performance for glucose detection under a low potential value of −0.4 V vs. Ag/AgCl (Table 2). A possible explanation for this behavior was the synergistic consequence of regular morphology and uniform isolated distribution. The authors stated that Au nanocubes/G composite film presents the possibility of being used in applications for trace analysis for new physiological activators by changing the corresponding proteins. The high catalytic activity and conductive capacities of the new composite material represent important properties that were demonstrated not only in biosensor construction but also offer the possibility of being successfully used in other studies closely related to electrocatalysis [91].

3.1.2 Hydrogen peroxide

The production of reactive oxygen species represents a process that can be associated with early signs of cancer or neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and multiple sclerosis. Precisely, reactive oxygen species appear as a consequence of changed cellular metabolism rising from a given disease state. These species are extremely reactive and induce redox reactions of cell structures leading to activation of immune response and apoptosis. Between these, hydrogen peroxide (H₂O₂) was intensively investigated as analyte to describe disease conditions. Easier H₂O₂ detection is as well a challenge in various domains like medicine, manufacturing, food production, and pharmaceuticals. Among the variety of possible applied techniques, electrochemistry consists of the simplest way of getting rapid and accurate results while demanding only a simple device, and it is realized by analyte oxidation or reduction (Figure 6) [21, 94, 95, 96].

Figure 6.

I vs. [H₂O₂] calibration curve recorded at (Fe-CA)-CPE (a) and undoped CA-CPE (b). Inset: amperograms recorded at an applied potential of 0.3 V vs Ag/AgCl, KCl_sat, and a rotating speed of 500 rpm, in 0.1 M phosphate buffer (pH 7) at (Fe-CA)-CPE (a) and CA-CPE (b) for consecutive addition of 1 mM H₂O₂. Reproduced from Ref. [21] with permission from the Elsevier.

By electrochemical reduction of GO-horseradish peroxidase (GO-HRP) to rGO-horseradish peroxidase (rGO-HRP) a new biocomposite was obtained by Selvakumar et al. [97]. The enzyme immobilization process was facile, and the obtained composite material was employed for the preparation of rGO-HRP modified screen-printed carbon electrode (SPE) in order to detect H₂O₂. The obtained sensor characteristics were a wide linear range, high selectivity, and good stability, which recommended its extended practical applications. The sensitivity value toward H₂O₂ reduction is presented in Table 3. The authors suggested that the fabrication process used for obtaining of rGO-HRP biocomposites can be utilized for the construction of G-based composite materials of a large range of electrochemically essential molecules with redox properties [97].

Electrodes	Analit	Linear range	Sensitivity	Detection limit	Ref.
Cu2O/GNs	Glucose	0.3–3.3 mM	—	3.3 μM	[52]
	H2O2	0.3–7.8 mM	—	20.8 μM
SPCE/rGO-HRP	H2O2	9–195 μM	0.09 A M^−1 cm^−2	—	[97]
H-GNs/AuNPs/GCE	H2O2	0.3–1800 μM	2.77 A M^−1 cm^−2	0.11 μM	[98]
H-GNs/GCE	H2O2	0.5–400 μM	—	0.2 μM	[99]
Au/G/HRP/CS/GCE	H2O2	5–5130 μM	—	1.7 μM	[23]
Pd/HRP/G	H2O2	25–3500 μM	0.092 A M^−1 cm^−2	0.05 μM	[17]
Hb/AuNPs/ZnO/rGO/GCE	H2O2	6–1130 μM	—	0.8 μM	[36]

Table 3.

Electroanalytical parameters of some G-based electrodes for H₂O₂ detection.

GCE—glassy carbon electrodes, CS—chitosan, His—histidine, AChE—acetylcholinesterase, GOD—encapsulated glucose oxidase, AuNPs—gold nanoparticles, PPy—polypyrrole, GDH—glucose dehydrogenase, PTZ-O—phenothiazone, PAMAM—poly(amidoamine), GNs—G nanosheets, HRP—horseradish peroxidase, SPCE—screen-printed carbon electrode, H—hemin, Hb—hemoglobin.

Elsewhere, an innovative biosensor based on GCE, which was modified with the AuNPs and hemin-G nanosheets (H-GNs), was successfully fabricated by Song et al. [98]. Their work demonstrated that the newly fabricated H-GNs/AuNPs/GCE bioelectrochemical sensor exhibited an improved electrocatalytic activity for H₂O₂ reduction process, when compared with the AuNPs/GCE, H-GNs/GCE, and bare GCE. The authors proved that the improved electrocatalytic signal was the result of two properties: (i) the improved specific surface area of the electrode surface, and (ii) the great loading of the H-GNs on the modified electrode surface. Therefore, an enhanced synergistic electrocatalytic influence was revealed concerning the AuNPs and H-GNs. Enhanced electroanalytical parameters of the developed biosensor (high value of sensitivity, wide linear response range, good stability and reproducibility, fast response time, and good analyte specificity) were the result of the new sensor material properties (Table 3) [98].

Another interesting research was done by Zhou et al., who achieved a new H₂O₂ biosensor based on the modification of GCE using G, chitosan CS, Au, and HRP [23]. They prepared individual G sheets by the insertion of ∙SO₃∙ radicals. By EDS (Energy Dispersive Spectroscopy) and TEM measurements, it was proved that the reduction and sulfonation techniques used for sensor material preparation did not produce the destruction of G morphology. As previously observed, the unaltered G structure is significant for the maintenance of the exceptional properties of G. By cyclic voltammetry techniques, the authors proved the existence of a direct electron transfer process among the fixed enzyme (HRP) and the surface of the electrode. Thus, a usual electrocatalytic reduction process of H₂O₂ occurs on the electrode surface. This work showed that in identical experimental conditions, the recorded current response of HRP/CS/GCE, Au/HRP/CS/GCE, sulfonated and reduced G/HRP/CS/GCE, and Au/sulfonated and reduced G/HRP/CS/GCE can significantly enhance the sensitivity of the developed biosensor due to the G structures presence (Table 3). It was also stated that a wide linear range, low detection limit, and long-term stability represent other excellent characteristics of this type of sensor [23].

The easy, low-priced, and highly sensitive and selective amperometric assessments employed for H₂O₂ and glucose detection, based on hemin functionalized G nanosheets (H-GNs), were developed by Guo et al. [99]. The obtained H-GNs hybrid nanomaterial combines the G nanosheet properties of high electrical conductivity and high surface area value with hemin properties (i.e., exceptional electrocatalysis and synthetic enzyme simulation). The study showed that the H-GNs are able to compete with the natural enzymes. The advantages of the obtained H-GNs are their facile synthesis method, the strength of materials, and the stability of materials in irregular conditions. Moreover, due to their excellent recorded results (Table 3), the obtained biosensor presents favorable possible applications in different fields, including clinical diagnostics, biotechnology, and chemical or pharmaceutical industry [99].

Another electrochemical biosensor for H₂O₂ selective detection was established by Nandini et al. [17]. The preparation method of this new electrode was realized by a co-deposition process of palladium and HRP, on the functionalized G-modified graphite surface electrode. The fabricated biosensor revealed an increased electrocatalytic activity concerning the reduction process of H₂O₂ at 0.02 V vs SCE. It was found that Pd, by its presence, decreases the over-potential of H₂O₂ reduction and grows the active surface area of the modified electrode. Furthermore, the HRP and Pd co-deposition escapes the poisoning occurrence of the modified graphite-based electrode. The sensor performance exhibited a fast response within less than 2 s and exceptional linear concentration range under determinate optimal experimental conditions (Table 3). For the acquired properties of the biosensor (i.e., selectivity, repeatability, feasibility and stability), acceptable results were obtained. The presented technique could be applied to produce biosensor for a wide range of applications [17].

Based on the hemoglobin (Hb) immobilized on an rGO, flower-like ZnO, and AuNPs nanocomposite modified GCE (AuNPs/ZnO/rGO/GCE), a new amperometric H₂O₂ biosensor was proposed by Xie and co-workers [36]. Each biosensor component was found to have an important contribution to the H₂O₂ reduction process. Therefore, the ZnO flower-like nanoparticles exhibit good biocompatibility and conductivity. Then, the ZnO-aminopropyl triethylene silane (APS)-AuNPs composite was found to have good uniformity and be suitable for protein attachment. rGO possesses high specific surface area and can increase the ZnO-APS-AuNPs composite conductivity and its mechanical resistance. The authors showed that by combining the advantages of each component used for bioelectrical sensor preparation (i.e., nanosized ZnO, AuNPs, and rGO), the Hb/AuNPs/ZnO/rGO/GCE), the obtained modified electrode can lead to acceptable sensors performances of high sensitivity, satisfactory construction reproducibility, and good storage stability. Their study has shown that AuNPs/ZnO/rGO/GCE amperometric third-generation biosensor offers an advantageous application for nanoparticles-based electrode materials in order to be employed in the investigation of direct electron transfer of proteins and the improvement of biosensors (Table 3) [36].

It is also important to reveal that the study performed by Liu et al., which focused on glucose detection and H₂O₂ reduction, demonstrated that with the non-enzymatic electrochemical sensor fabricated from G nanosheet-wrapped Cu₂O nanocubes (Cu₂O/GNs), good sensor characteristics for glucose oxidation can be obtained [52]. Also, they proved that Cu₂O/GNs hybrids can be produced by an easy process at low temperature and that they exhibit excellent electrocatalytic activity toward H₂O₂ reduction. Therefore, they tested the electrochemical detection capacity of glucose and H₂O₂ (Table 3). The developed sensor revealed good performances. An explanation for this behavior can be the enhanced electrocatalytic surface area of the electrode and the high conductivity of the G-hybrid nanostructure, reflected in the improved electron transfer ability through this matrix. Additionally, the authors stated that their synthesized Cu₂O/GNs structure revealed enhanced electrochemical stability in contrast with the Cu₂O nanocubes alone due to the G nanosheets that covered the oxide nanocubes. The advanced Cu₂O/GNs nanocomposite material opens up new opportunities for applications in several varieties of biosensors, bioelectronic devices, and catalysts [52].

Some advances in in vivo electrochemical analysis of H₂O₂ were reported by Deng and co-workers. Their interest was directed toward the electrochemical redox process of H₂O₂ at the electrode surface and on the diversity of catalysts (synthetic electrocatalysts or biomolecular electrocatalysts) used for improved electrochemical analysis of H₂O₂. A higher selectivity and sensitivity of the proposed biosensors were followed. The new technique, the photoelectrochemical (PEC) method for H₂O₂ detection was also discussed. The developments in the high selectivity analysis of H₂O₂ at the cellular and in vivo levels was highlighted [30]. Another review focused on the G as electrode materials for (bio)sensors applications was written by Shao et al. [32], who demonstrated the performances for hydrogen peroxide, NADH, dopamine, DNA, or heavy metals detection.

An important work dedicated to the significance of flexible sensors on H₂O₂ detection was realized by Giaretta et al. [87]. They compared the electroanalytical parameters for H₂O₂ detection obtained on different biosensors based on various material substrates. It was concluded that the carbonaceous materials are better in comparison with the polymeric substrates, which are the cheapest. The carbon materials-based biosensors results were based on the corroborated effect of the increased electrical conductivity, increased permeability, and increased porosity, respectively.

Different types of materials (noble metals, metal oxides, polymers, carbon materials, and other two-dimensional materials) employed in sensor development for H₂O₂ detection were presented in the review work of Yu and co-workers [100]. Additionally, their work presents the challenges and future prospects in the biological applications of electrochemical sensors for H₂O₂ detection.

3.1.3 Neurotransmitters and neurochemicals

Neurotransmitters are endogenous compounds that permit the transmission of nerve impulses between two neurons or between neuron and effector, named ‘target’ cell. Thus, the nervous system is based on the role of chemical couriers of the neurotransmitters, which transfer the information across the synapses by excitation or inhibition of the subsequent neural or effector cell. Neurotransmitters are ordered into monoamines (histamine, adrenaline, dopamine (DA), noradrenaline, serotonin, and melatonin), amino acids (aspartate, D-serine, glutamate, gamma-aminobutyric acid, and aminoacetic acid (glycine)), peptides (somatostatin, cocaine, and opioid), and other (including acetylcholine, adenosine, anandamide, and nitrogen monoxide) [101].

3.1.3.1 Dopamine, ascorbic acid, uric acid

It is well-known that monoamine neurotransmitters have in their structure one amino group connected by a chain of two atoms of carbon (∙CH₂∙CH₂∙) with an aromatic ring. These types of neurotransmitters have a significant importance in secreting and producing neurotrophins via astrocytic glial cell. It is an essential local cellular source of trophic support, present both in healthy and unhealthy brain. Growth of neutrophins stimulates the survival of neurons and is known as neurotrophic factors. Based on the neuron behavior, the neurotransmitters act antagonistically, namely neurotransmitters that play an inhibitory role to relax the brain and those that play an excitatory role in stimulating the brain [102]. Dopamine (DA) owns both excitatory and inhibitory classification, being thus a unique neurotransmitter. DA vital roles lie in adjustable attention, motor control, cognition, executive functions, pleasure, motivation, arousal, reinforcement, reward, and hormonal processes. Also, dopamine is extensively dispersed in the main systems of the human body, such as central nervous, renal, hormonal, and cardiovascular.

Neurological problems in the human body can be due to the anomaly in the amount of dopamine. Thus, the illness such as Parkinson’s disease (degenerative disorder of the central nervous system that mainly affects the motor system), restless leg syndrome (like Parkinson’s disease, this is another long-term disease that manifests itself through the uncontrolled movement of the legs), attention deficit hyperactivity disorder (ADHD) (characterized by lack of ability to concentrate, hyperactivity and impulsivity), schizophrenia (a disease in the psychiatric spectrum in which episodes of psychosis occur), and infection with human immunodeficiency viruses (HIV) (a virus that over time can cause AIDS—the gradual collapse of the immune system leading to the onset of opportunistic infections and cancers) are strongly associated with a low level of dopamine. Also dopamine is also greatly correlated with the reward mechanism in the brain. On the other hand, the consumption of prohibited drugs or substance abuse leads to an increase in dopamine levels. Thus, the forbidden substances such as heroin or cocaine, and not compulsory substances such as nicotine or alcohol, block the DA carrying that inhibits the reuptake of dopamine. As was shown before, dopamine, a neurotransmitter, which is vital for message transfer functions, produces, in this case, an amplified risk of depression and drug dependence. Thus, the discovery of a reliable analytical technique is significant and necessary in order to estimate the disease evolution.

A simple and “green” technique to produce G flowers that were exploited to modify carbon fiber electrode (CFE) in order to detect AA, DA, and UC was employed by Du et al. [34]. The G flowers, well deposited on the surface of CFE, detect separately electroactive compounds as AA, DA and UC. Besides, this study proved that the obtained G flowers based modified electrodes, can detect simultaneously AA, DA and UC, with distinct signals from each other. Thus, great electrocatalytic activities of the electrode material toward the oxidation process of electroactive AA, DA and UC, good selectivity and sensitivity, were demonstrated (Table 4). Excellent performance was obtained with the modified electrode when the detection of real samples was proposed. The obtained results offer appreciable evidence for the use of G as a sensitive modifier material electrode in order to detect electroactive biomolecules [34].

Electrodes	Analyte	Linear range	Sensitivity	Detection limit	Ref.
GEF/CFE	AA	73.52–2305.53 μM	0.011 A M^−1	73.52 μM	[36]
	DA	1.36–125.69 μM	1.02 A M^−1	1.36 μM
	UA	3.98–371.49 μM	0.074 A M^−1	3.98 μM
SGGT	DA	10 nM–1 μM	—	1 nM	[103]
	AA	1 μM–100 μM	—	1 μM
	UA	10 μM–100 μM	—	10 μM
NG	DA	0.1–0.45·mM	0.044 A M^−1	0.93 μM	[53]
MIPs-G/CS	DA	1–100 nM; 0.1–100 μM	188 A M^−1; 0.94 A M^−1	10 pM	[44]
ZnO NWAs/3D-GF	UA	0.5–40 μM	4.25 A M^−1	1 nM	[51]
	DA	0.5–40 μM	4.81 A M^−1	1 nM
	AA	0.5–80 μM	0.38 A M^−1	—

Table 4.

Electroanalytical parameters of some G-based electrodes for ascorbic acid, uric acid, and dopamine detection.

CS—chitosan, AA—ascorbic acid, DA—dopamine, UA—uric acid, GEF/CFE—G flowers/carbon fiber electrode, SGGT—solution-gated G transistor, NG—nitrogen-doped G, MIP—molecularly imprinted polymer, ZnO NWAs/3D-GF—nanowire arrays fabricated on 3D G foam.

Using a solution-gated G transistor (SGGT) with a G gate electrode, a very sensitive dopamine electrochemical sensor was achieved by Zhang et al. [103]. The detecting mechanism of the obtained system was ascribed to the electrooxidation process of DA at the gate electrode. This electrode changed the potential distribution at the boundaries between the G gate electrode and the G channel. A perfluorinated membrane with ionic properties and excellent selectivity for dopamine was obtained after the addition of a thin layer of Nafion to G gate electrode. The obtained sensor showed a limit of detection for dopamine down to 1 nM. This represents a worthy result for dopamine detection in medical uses. The dopamine interference measurements with AA and UA showed good selectivity of the device, proved by a recorded signal up to four orders of magnitude lower when compared with that obtained for dopamine. Since the channel and the gate of the device are both made of G, they have the advantage that they can be produced on different substrate materials (containing flexible, elastic, pliable, and stretchy ones) at low temperatures by suitable procedures. Centered on the equivalent mechanisms, various additional types of biosensors could be imagined and designed in the following years, and the whole-G SGGT represents a good solution for one-use, bendable, and very sensitive biosensors [103].

Li and co-workers demonstrated that nitrogen-doped rGO (N-rGO) with a very porous matrix and adjustable structure can be produced in three steps: first, the molecular functionalization; second, the fast thermal expansion–exfoliation, and third, the covalent binding [53]. The nitrogen-doped configurations, controlled by varying the temperature of expansion–exfoliation process, were used for the preparation of screen-printed electrodes. Judging by the potential value of the peak current recorded to the oxidation process of AA, DA, and UC among the nitrogen-doped sample, the pyrrolic-N revealed the maximum electrocatalytic activity. Nevertheless, the corresponding peak currents, of the oxidation of AA, DA, and UC, are related to the corroborated effect of the nitrogen-doped sample distribution, and structural properties (specific surface area and porosity) and the electroanalytical parameters presented in Table 4. The prepared SPEs exhibited high peak currents (for biomolecules’ electrooxidation process), good selectivity (good peak separation), and sensibility for the detection of AA, DA, and UC from a blend [53]. A molecularly imprinted polymers (MIPs) based on Chi–G composite, as the functional matrix, was used by Liu et al. to develop a sensor for DA electrochemical detection [44]. Thus, the obtained MIPs-GR composite was used to modify GCE, in order to fabricate the sensor (MIPs-G/GCE). The improved sensitivity and low value of the detection limit of MIPs-G/GCE sensor for DA oxidation (Table 4) can be explained based on the special characteristics of G. The selectivity, stability, and reproducibility remained just as for the MIPs sensor. It was stated that the achieved information could offer a possible rapid and reliable method for DA determination in biological samples [44].

Another significant G-based sensor for dopamine was produced by Yue et al. [51]. Thus, the detection of three biomolecules (UA, DA, and AA) was realized by employing 3D G foam containing ZnO nanowires, vertically arranged at the electrode surface. Differential pulse voltammetry (DPV) technique was used for UA, DA, and AA electrochemical detection. The new structural design combined (i) the large mesoporous surface area 3D G structures that facilitated easy diffusion of ions through the electrode material, with (ii) the increased conductivity of 3D G foam, which led to a good electron transfer process, and (iii) the active sites of ZnO nanowires, which assure a high selectivity. The present study proved that the UA, DA, and AA selectivity was the result of thermal annealing of ZnO surface.

A high selectivity and a low value of detection limit for UA and DA were obtained with the optimized ZnO nanowire/3D G foam electrochemical sensor (Table 4). The obtained results were clarified by the gap variance among the LUMO (lowest unoccupied molecular orbitals) and HOMO (highest occupied molecular orbitals) of a biological molecule for a set of specified electrodes. For the Parkinson’s disease test, the UA level was 25% lower than in healthy individuals. It was concluded that the reported work can open new perspectives for UA application as a biomarker for Parkinson’s disease, which can offer better medical diagnostic control in addition to the possibility of tracking the disease [51].

3.1.3.2 Serotonin

Several chemically different types of G nanosheets were synthesized by Kim et al. with the aim of using them as electrocatalysts for serotonin (5-hydroxytryptamine, 5-HT) [24]. In order to estimate the G nanosheets surface morphologies, X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FE-SEM) techniques were used. By electrochemical impedance spectroscopy (EIS) technique the electrocatalytic activity was investigated. In this study, the three different arrangements of rGO obtained did not reveal some significant changes in the obtained XPS spectra and FE-SEM images but showed dissimilar electrochemical performance. Thus, EIS recorded spectra exhibited a dissimilarity in the electron transfer resistance. This result is in good agreement with the reducing agent. The obtained parameters for 5-HT determination and the acquired EIS results were in agreement as well. The rGO-based GCE sensor obtained for 5-HT detection exhibited high sensitivity, good selectivity, and smaller electron transfer resistance compared with previously obtained sensors. Between the evaluated G-modified GCEs, the best electrochemical sensor properties (i.e., lowermost detection limit, uppermost sensitivity and selectivity, broadest linear range, fastest response time, alongside the greatest defined peak of 5-HT) were obtained for rGO reduced using hydrazine and ammonia solution (Table 5). This study proved an irreversible diffusion-controlled electrode process for a 5-HT electrooxidation reaction mechanism [24].

Electrodes	Analyte	Linear range	Detection limit	Ref.
rGO/PANI	Serotonin	0.2–10.0 M	11.7 nM	[37]
GO-S-(CH2)4-SH/GCE		1–100 μM	16 μM	[24]
GO-SH/GCE			0.38 nM
rGO3/GCE			0.52 nM
GO/GCE			18 μM
G/Au/GCE	Epinephrine	0.05–8.0 μM	7 nM	[25]
rGO/Thi/AuNPs	CEA	5.55 × 10^−5–1.66 nM; 1.48 × 10^−4–4.44 nM	3.61 fM	[38]
rGO/PB/AuNPs	AFP		13.11 fM
G/MBs–Ab1/CEA/Ab2–AuNPs–HRP	CEA	27.7–33.3 pM	27.7 pM	[39]

Table 5.

Electroanalytical parameters of some G-based electrodes for different types of analytes.

GCE—glassy carbon electrodes, GO—graphene oxide, rGO—reduced graphene oxide, AuNPs—gold nanoparticles, PANI—polyaniline, CEA—carcinoembryonic antigen, MBs—magnetic beads, Ab—antibody, AFP—α-fetoprotein, PB—Prussian Blue, Thi—thionine.

A two-layered membrane sensing interface for serotonin detection was constructed by Xue et al. [37]. The production of the obtained device was based on the use of nanosized rGO/polyaniline (PANI) composites and molecularly imprinted polymers (MIPs) surrounded with AuNPs (AuNPs@MIPs). With the aim of obtaining a good sensitivity and selectivity of the elaborated device, the rGO/PANI nanocomposites were produced by the electrodeposition method. First, the protonated anilines were anchored by electrostatic adsorption on the rGO sheets. Afterward, the rGO/PANI nanocomposite film was made by cyclic voltammetry process on the surface of bare GCE. Over the nanosized rGO/PANI composites membrane, the AuNPs@MIPs were deposited. Interestingly, the obtained material interface showed improved properties for (i) selectivity to 5-HT, (ii) electric conductivity, and (iii) electrocatalytic activity (Table 5). The new AuNPs/PPy/RGO/GOD/chitosan-modified electrode was efficaciously applied toward 5-HT detection in human serum specimens. In the meantime, the interferences produced from AA, DA, UA, and epinephrine (EP) did not affect the 5-HT detection. Therefore, the approach was recommended by this research team for a sensitive and selective detection of targeted biomolecules in real sample [37].

3.1.3.3 Epinephrine

Adrenaline or epinephrine, manufactured by the suprarenal glands and certain neurons, is a hormone neurotransmitter. It is also a medication and fulfills a pivotal function in the fight-or-flight reaction. Thus, under its action, the blood circulation to muscle tissues, cardiac output, pupil dilation, and blood sugar increase. This happens because of the adrenaline binding to alpha and beta receptors. Due to its importance, many research works were focused on this subject [18, 25].

In order to prepare a sensor for epinephrine (EP), Cui et al. started from a method based on chemical reduction of both Au (III) and GO, to prepare rGO/Au nanocomposites [25]. Then, the prepared rGO/Au nanocomposites, used as modifier-sensitive material for GCE surface (rGO/Au/GCE) were found to confirm an improved electrochemical activity toward EP (Table 5). The role of gold nanoparticles (nano-Au) included in rGO matrix was that of a spacer in order to avoid the rGO sheets aggregation. The achieved rGO/Au/GCE proved high sensitivity for the detection of EP. The recorded CV in the presence of ascorbic acid (AA), which show total peak separation between EP and AA, demonstrated good electrochemical sensor selectivity. Furthermore, rGO/Au/GCE revealed exceptional electron transfer capacity and exceptional electrocatalytic ability to additional biomolecules, including DA, AA, NADH, and pyrocatechol, opening up, thus, new perspective for applications improvement of rGO/Au nanocomposites in (bio)sensors [25].

3.1.4 Cancer and disease biomarkers

Cancer biomarker detection represents one of the most important achievements in the field of biosensor production. An interesting review work was done by Alsharabi et al., who highlighted the capacity of G and its various derivatives to conjugate their unique chemical structure and characteristic optical, electronic, mechanical, and thermal properties. These special materials may represent an excellent sensing platform for cancer biomarkers detection, representing a possible response to the important challenge in the diagnosis of cancer at the initial stages [29]. The G-based electrochemical biosensors for cancer biomarker detection were fabricated by using G surface that was improved with magnetic beads and enzyme-marked antibody-AuNPs [39]. One should emphasize that the development of an electrochemical biosensor for cancer biomarker determination remains essential for early discovery and diagnosis. In addition, the electrochemical sensor presents the advantages of improved characteristics (i.e., rapid, precise, and sensitive) in comparison with other investigation techniques.

In order to prepare a new biosensor for cancer-related biomarker, Jin and co-workers used a G platform that was prepared by CVD. Then, the MBs and enzyme-marked antibody-AuNP bioconjugate were added [39]. The attachment of MBs, covered with capture antibodies (Ab1), to the G sheet surface, was realized by applying an external magnetic field, with the aim to avoid the reduced G matrix conductivity. With the aim of increasing the sensitivity of the multi-nanomaterial-built biosensor, the AuNPs were improved with HRP and detection antibody (Ab2), forming the conjugate Ab2–AuNPs–HRP. In this electrode arrangement, the fast electron transfer between the multi-nanomaterial present on the electrode surface and analyte target was realized, and a low value for carcinoembryonic antigen detection limit was reached (Table 5). The acquired results evidenced a fast response and recovery time, which was more improved compared to that achieved when the old-style approaches were used. The good sensors achieved properties (sensitivity, specificity, simplicity of construction technique, ease of use, fast analysis, and reusability) confirm that the developed biosensor can be used for cancer’s medical diagnosis [39].

A label-free electrochemical multiplexed immunosensor based on G nanocomposites was fabricated for the recognition of both carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) by Jia et al. [38]. The indium tin oxide (ITO) sheets were used to be modified with anti-AFP fixed on G nanocomposite matrix. The electrode fabrication and voltametric sensing technique were centered on the electron transfer delay determined by the engineered antibody-antigen immunocomplex present on the ITO electrode surface. The obtained multiplexed immunosensor facilitated the concomitant detection of both CEA and AFP, and the found linear ranges are presented in Table 5. The limit of detection value for CEA and for AFP, are depicted in Table 5. A few aspects were pointed out as follows: (i) the fabricated immunosensor escaped the marking of both antigens or antibodies, making it easier and preventing the cross-talk among diverse analytes; (ii) G nanocomposites used as supporting scaffold were synthesized by a facile route, and the shapes and quantity of the immobilized AuNPs, by this method, could be easily controlled; (iii) the immunoassay having a worthy stability, large linear ranges received a good correlation with ELISA (enzyme-linked immunosorbent assay) and could be used in medical diagnosis. It was also stated that this simple approach could be adapted and combined for new biosensor applications [38].

4. Conclusions

Since its discovery, G has been tested in a large diversity of biosensing applications owing to its remarkable electrical, mechanical, and optical properties as well as its unique structure. If one compares G-based biosensors with conventional ones, clear benefits, such as high sensitivity and selectivity, low detection limit, reproducibility, stability, fast response, or easy miniaturization, make G-based biosensors a real candidate for a novel and efficient class of biosensors for medical applications.

Electrochemical biosensors offer an inexpensive, facile, fast, sensitive, and selective detection of biomolecules, and the use of G and its composites for biosensors development is due to its unique features combined with some peculiar properties. Moreover, the reusability of the biosensors is aimed. The present chapter summarized the G-based electrochemical sensors developed for sensing biomolecules and highlighted their significant advances. Thus, G and GO present a wide range of electrochemical potential and fast electron transfer rate. The drawback of using G in biosensors construction is its possible toxicity, as stated in the international nanotechnology guidelines. On the other hand, G can be easily functionalized/modified by electrodeposition, polymerization, electrochemical doping, or other methods, and even if G could be cytotoxic, biomedical applications can benefit from non-toxic, biocompatible and water-dispersible G layers obtained by chemical functionalization with different ligands. By biosensors incorporation into strong, transportable, and miniaturized devices, the detection of biomolecules and toxins for usability in clinical and diagnostic fields can be achieved.

Although there is a massive investment both in academia and industry, G-based biosensors are still at an incipient level, and commercial biosensors are yet to come. Nevertheless, there is a slow but promising translation into medical applications.

Acknowledgments

This work was supported by a grant from the Ministry of Research, Innovation, and Digitization, CNCS/CCCDI—UEFISCDI, project number PN-III-P2-2.1-PED-2021-3156, within PNCDI III.

List of abbreviations

AA	ascorbic acid
Ab	antibody
AChE	acetylcholinesterase
ADHD	attention deficit hyperactivity disorder
AFP	α-fetoprotein
APS	aminopropyl triethylene silane
CDH	cellobiose dehydrogenase
CEA	carcinoembryonic antigen
CS	chitosan
CVs	cyclic voltammograms
CVD	carbon vapor deposition
DA	dopamine
DC	direct current
DNA	deoxyribonucleic acid
DPV	differential pulse voltammetry
EDS	Energy Dispersive Spectroscopy
EIS	electrochemical impedance spectroscopy
ELISA	enzyme-linked immunosorbent assay
EP	epinephrine
FAD	flavin adenine dinucleotide
FE-SEM	field emission scanning electron microscopy
FGO	functionalized graphene oxide
G	graphene
GCE	glassy carbon electrodes
GDH	glucose dehydrogenase
GEF/CFE	G flowers/carbon fiber electrode
GO	graphene oxide
rGO	reduced graphene oxide
GOD	glucose oxidase
GNs	G nanosheets
H	hemin
Hb	hemoglobin
His	histidine
HIV	human immunodeficiency viruses
HOMO	highest occupied molecular orbitals
HRP	horseradish peroxidase
HR-TEM	High Resolution – Transmission Electron Microscopy
H-GNs	hemin-G nanosheets
ITO	indium tin oxide
LUMO	lowest unoccupied molecular orbitals
MBs	magnetic beads
MIPs	molecularly imprinted polymers
NADH	nicotinamide adenine dinucleotide reduced
NG	nitrogen-doped G
NPs	nanoparticles
NWAs/3D-GF	nanowire arrays fabricated on 3D G foam
PAMAM	poly(amidoamine)
PANI	polyaniline
PB	Prussian Blue
PEC	photoelectrochemical
PPy	polypyrrole
PTZ-O	phenothiazone
SEM	Scanning Electron Microscopy
SPE	screen-printed electrode
SPCE	screen-printed carbon electrode
SGGT	solution-gated G transistor
UC	uric acid
Thi	thionine
TEM	Transmission Electron Microscopy
XPS	X-ray photoelectron spectroscopy
5-HT	5-hydroxytryptamine-serotonin