Overview of enzyme-contained and non-enzymatic graphene paper/film sensors for detection of glucose.
Abstract
Graphene paper as a new form of graphene-supported nanomaterials has received worldwide attention since its first report in 2007. Due to their high flexibility, lightweight and good electrical conductivity, graphene papers have demonstrated the promising potential for crucial applications in electrochemical sensors and energy technologies among others. In this chapter, we present some examples to overview recent advances in the research and development of two-dimensional (2D) graphene papers as new materials for electrochemical sensors. The chapter covers the design, fabrication, functionalization and application evaluations of graphene papers. We first summarize the mainstream methods for fabrication of graphene papers/membranes, with the focus on chemical vapour deposition techniques and solution-processing assembly approaches. A large portion of this chapter is then devoted to the highlights of specific functionalization of graphene papers with polymer and nanoscale functional building blocks for electrochemical-sensing purposes. In terms of electrochemical-sensing applications, the emphasis is on enzyme-graphene and nanoparticle-graphene paper-based systems for the detection of glucose. We finally conclude this chapter with brief remarks and outlook.
Keywords
- graphene paper
- 2D-layered nanomaterials
- electrochemical sensor
- metal oxide
- polymer
- nanoparticle
1. Introduction
Precise monitoring of chemical or biological processes is of extreme importance for medical and biological applications. Electrochemical sensors can ideally fulfil that goal by converting a chemical or a biological response into a processable and quantifiable signal. In the past two decades, intensive research and development of electrochemical sensors have enabled to fabricate different types of devices. After the development of many successful commercial electrochemical sensors in the classic configurations, currently there is a notable transition and increasing demands for the development of flexible and wearable sensors. The development of flexible electrochemical sensors depends crucially on the discovery and preparation of free-standing and flexible new materials.
Flexible electronic devices, especially flexible electrochemical sensors, have become an advanced technology with the aim at solving some tremendous real-world challenges, such as in situ sensing for health and environmental problems. Compared with conventional devices, flexible electrode-based devices are versatile and highly adaptive. For example, such devices occupy less space and could be compatible with any shape-targeted systems such as human body and rough or irregular-shaped substrates. Ideal flexible electrodes should provide high conductivity for sensing electrons and thermal energy, high stability and excellent mechanical strength. In this regard, carbon nanotube (CNT)-based devices had dominated research and development attention before [1, 2], because of the unique one-dimensional nano-channel mechanical strength. However, one has come to realize that CNT-based flexible electrodes are largely limited to fundamental interests because of several serious technical challenges, such as high cost of producing CNTs, complex process of obtaining high-density vertically aligned CNTs, chemical and biocompatibility concerns and difficulties in achieving large-scale production, which can hardly overcome. On the other hand, the recent rise of synthesis and processing of two-dimensional (2D) graphene and its derivatives offer a new and promising opportunity for developing a novel class of flexible electrodes most likely with required physiochemical properties. Graphene paper, as one of the derivatives from graphene or graphene oxide (GO)-supported building blocks, was introduced by Ruoff and co-workers in 2007 [3]. Due to their remarkable mechanical property, tunable conductivity and versatile functionality, graphene papers could promote the research and development of new-generation flexible electrode-based sensors and other electronic devices. This chapter aims at highlighting some recent examples using graphene papers as electrode materials for developing flexible and ultrasensitive electrochemical sensors.
2. Synthesis of graphene paper-based electrodes
Thanks to the intrinsic planar structure of graphene sheets, it is feasible to effectively control the periodic alignment of graphene nanosheets into 2D graphene papers/films/membranes through ionic interaction, hydrophobic effect, hydrogen bonding and/or π-π stacking [4, 5]. The resulting 2D graphene materials not only retain some of the key properties of individual graphene sheets but also create new or collective properties resulting from their unique structure. Moreover, a wide range of functionalized materials, such as metal, polymer, metal oxide (MO) and other semiconductors, can be loaded into 2D-layered GO papers, endowing the graphene papers with multiple functionalities required by specific applications [4–8]. Given the excellent physicochemical properties of graphene paper and tunable functional groups, these 2D architectures have been widely studied for their applications in water purification, biomimetics, sensors, flexible electrodes, energy conversion and storage and optoelectronic devices [7–11]. As outlined below, several crucial methods have been developed to assemble the layered structure of graphene papers.
2.1. Chemical vapour deposition
Chemical vapour deposition (CVD) is a chemical process used to produce high quality, high performance and solid materials. The CVD technique is a direct and promising method for the preparation of large-scale, near-perfect graphene films on various substrates. The graphene films obtained by CVD methods displayed high flexibility, transparency and electrical conductivity [12, 13], which is attractive for various electronic devices. Commonly, transition metals, such as Ge [14, 15], Ni [16, 17], Cu [18, 19], Rh [20–22] and Co [23, 24], are used as the substrates for the CVD growth of graphene films. Due to very low carbon solubility in Cu, ease of etching and feasibility of high-quality graphene transfer, Cu has emerged as a favoured substrate chosen mostly for the growth of large-area graphene films [25], which was first introduced by Ruoff’s research group in 2009 [13]. Large-area and single-layer graphene films with a low percentage (less than 5%) of the areas having few layers can be deposited on copper substrates by CVD process using methane as a carbon source (Figure 1a). The graphene film was robust enough to be transferred onto Si/SiO2 substrates for fabrication of dual-gated field-effect transistors. They also demonstrated that as-synthesized graphene films could be transferred to other substrates (Figure 1b and c). To meet some special requirements, non-metal materials, such as Si [14, 15], SiO2 [26–28], BN [29, 30] and Si3N4 [31, 32], were also used as substrates. However, the non-metal substrates showed the drawback limitations including slow growth rate and discontinuous size. For the CVD growth of graphene films on a metal substrate, it is necessary to mention that a transfer step for the following integration of graphene film into any solid-state electronic devices is usually required. Given the polycrystalline nature of the film and roughness of metal foils, industrial roll-to-roll transfer technology could be employed. Moreover, free-standing and element-doped (e.g. N and S) [33] graphene films can also be prepared directly by the CVD technique. However, the conventional etching method could be satisfied at laboratory scales, but it is very challenging for the large-scale preparation of high-quality graphene films arising from high-handling skills and time-consuming setback.
2.2. Solution-processed assembly
Owing to oxygen containing polar functional groups existing on the surface and at the edges, GO is well dispersible in many polar solvents including water [34]. This structural feature and chemical capability facilitate GO as a favourite starting material for the assembly of various graphene architectures such as graphene papers and three-dimensional (3D) graphene sponges.
2.2.1. Vacuum filtration
To build a well-organized 2D macroscopic structure using single-layer GO sheets as a start material, Ruoff’s group introduced a facile method for the fabrication of graphene paper. That is, GO sheets could be assembled into a paper-like material under a directional flow. Vacuum filtration of colloidal dispersions of GO sheets through an anodic membrane filter yielded free-standing GO papers with tunable thickness and mechanical property in a dried state. The fracture edges of a GO paper sample were imaged by scanning electron microscopy (SEM) to reveal its well-ordered layers over almost the entire cross section of the paper sample. The sandwiched structure between less densely packed ‘wavy’ skin layers was approximately 100–200 nm thick. In a typical GO paper specimen, the layer-to-layer distance (d-spacing) was measured as approximately 0.83 nm by the X-ray diffraction (XRD) spectrum [3]. As a result of its facile processability, vacuum filtration has been popularly used to prepare free-standing graphene papers. For example, cellular graphene paper was prepared by the following three steps [35].
2.2.2. Layer-by-layer assembly
Self-assembly is a popular approach towards cost-effective preparation of thin films, which has also been applied for fabricating multilayer graphene papers with tunable composition and architecture [8, 10, 38]. The driving forces involved in layer-by-layer (LBL) assembly generally include hydrogen bonding, electrostatic interaction and covalent bonding [8, 39, 40]. Zou and Kim reported a diffusion-driven LBL assembly process to prepare graphene-based architecture and demonstrated its application for the construction of GO sheets into various 3D macrostructures [41]. This assembly process is driven by the complexation of the negatively charged GO sheets and positively charged branched polyethylenimine (b-PEI) at a defined interface. The key step for assembling GO sheets to GO paper is that the diffusion of b-PEI molecules allows the complex to continuously grow into foam-like frameworks with tunable porosity. In a typical experimental process as shown in Figure 3, a small amount of the b-PEI solution was
2.2.3. Other solution processing methods
Besides the methods mentioned above, solution casting, spin coating, spray coating and dip coating [42–45] have also been explored for the preparation of graphene papers or films. For example, GO or rGO solution was deposited on poly(ethylene terephthalate) (PET), SiO2/P++Si and Au via various techniques to prepare thin films [46–49], although it is still a challenge to obtain graphene films with uniform thickness and few wrinkles by such type of approaches.
2.3. Electrophoretic deposition
Common synthetic approaches for the fabrication of graphene papers often use harsh chemicals and require complex pathways. Furthermore, the subsequent transfer process of the graphene paper onto a specific substrate even makes the overall process more complicated and time-consuming. In contrast to these concerns, electrophoretic deposition (EPD) process is useful for applying materials to any electrically conductive surface. With the advantages of low cost, easy handling and suitability for industry-scale production, EPD has been an effective technique to deposit graphene on conductive substrates for a wide range of applications [50]. A number of reports have demonstrated the deposition of GO on various substrates for the fabrication of suitable electrodes via EPD process, such as Cu and Ni foams, ITO, stainless steel and Pt [50, 51]. As a representative example, Liu et al. developed a two-step procedure, which involved EPD of GO and its subsequent transformation of GO to rGO by in situ electrochemical reduction. More recently, a single-step preparation procedure of metal-supported rGO film was reported. A schematic of the electrochemical setup used for the preparation of stainless steel-supported rGO is shown in Figure 4 [52]. The wireless graphite was oxidized into a colloidal dispersion of GO due to the effect of an electric field, and GO nanosheets migrated electrophoretically towards the anodic side of the electrochemical cell where they were deposited by van der Waals forces in the form of rGO film (
2.4. Other methods
In addition, a number of other approaches have also been reported for constructing graphene papers/films with hierarchical structures and high electrochemical or physical performances. For instance, Cao et al. constructed graphene films with high conductivity and mechanical stability via reduction-oxidation reactions between GO and active-metal substrates [53]. Honeycomb-structured graphene films were prepared by the template method [54, 55]. Bubble-like structured graphene film was also fabricated using monodispersed poly(methyl methacrylate) (PMAA) latex spheres as the sacrificial templates [56].
3. Structural and morphologic characterization
As-synthesized graphene papers are a very promising candidate for energy device and sensor applications. Structural characterization is of paramount importance for understanding the correlation between their nanostructures and performances. Therefore, some advanced techniques are intensively used to characterize graphene paper-based materials, such as atomic force microscopy (AFM), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX) and thermogravimetric analysis (TGA). In particular, electronic microscopies (SEM and TEM) are among the most powerful techniques to reveal the structural details of nanostructured graphene papers. For example, a combination of TEM and SEM was used to systematically characterize Au@PBNPs hybrid graphene papers. It is shown that Au@PB NPs were well distributed on graphene sheet surface (Figure 5), and cross-sectional SEM images indicated that the Au@PB NPs were successfully doped into the interlayers of the graphene paper to form a layer-by-layer sandwiched structure (Figure 6) [37].
4. Functionalization of graphene papers
The preparation and applications of graphene paper-based materials are attracting huge interests due to their unique electronic, optical, magnetic, thermal and mechanical properties. Compared to pure graphene papers, functionalized graphene papers or membranes can offer notable advantages ascribed to their hybrid structure, specific functionality and improved physicochemical properties. In general, graphene papers could be functionalized with a variety of chemical components such as metallic materials, metal oxides, polymers and supramolecular units, via various methods. Here, we only focus on three types of functional derivation, that is, metallic nanostructures, metal compounds and polymers, which are in favour of electrochemical-sensing applications.
4.1. Metallic nanostructures
Nanostructured metals are attracting intensive interests from the scientific community, owing to their fabulous properties and diversity of applications [57, 58]. A number of studies have explored the cooperation and synergistic effects between nanostructured metals and graphene paper, such as Pt nanoparticles (PtNPs) [59], Pt nanowires (PtNWs) [60], Au@Pt core-shell NPs [61], Au nanoparticles (AuNPs) [62] and Ag nanoparticles (NPs) [63]. As we mentioned in Section 2.1, CVD has made a great contribution to the preparation of graphene films. Hydrocarbon gases, through CVD consolidating on metal surfaces, enable the formation of a uniform and large-size graphene film, which can then be transferred onto solid substrates decorated with metallic NPs [64]. For example, Du and co-workers fabricated an AuNP-modified graphene film by a CVD-thermal release tape method, which has been used as an active substrate for the surface-enhanced Raman scattering (SERS) detection of analytes [65]. Furthermore, to enhance the electrical properties and surface plasmon signal of the graphene film, Ag nanowire was deposited further on the graphene film by a two-step procedure. In general, graphene films were
Self-assembly provides a simple and time-saving way to prepare large-scale nanostructure-modified graphene paper by precisely controlling the experimental parameters. For example, AgNP-graphene hybrid paper was assembled through electrostatic self-assembly.
4.2. Metallic compound nanostructures
Metallic compounds, especially transition metal oxides (TMOs) or transition metal hydroxides (TMHs), are a key family of materials in a variety of current demanding needs for sensor, catalysis, energy storage and conversion, optical electronics and piezoelectric mechanics, attributed to their nature of versatile functionality [76]. Given such a fact, metallic compound-functionalized graphene papers have been a hot topic in recent years. Commonly, metal oxide and GO were synthesized separately, and then MO-graphene paper was fabricated through mixing the two (or more) components and applying a typical vacuum filtration process. For example, our group has reported the fabrication of free-standing graphene–
In addition, non-metallic nanostructures such as those carbon-containing building blocks (e.g. CNTs, fullerenes, carbon black, carbon fibre and carbon spheres), Si, SiO2, Si3N4 and others [7, 10, 51] were also widely used to functionalize graphene papers.
4.3. Polymers
Besides inorganic nanocomposites, polymer-functionalized graphene papers also have widely been used for sensor and energy conversion and storage applications. In general, the polymer could be stabilized with graphene paper through the two approaches: (1) covalent bonding. Because GO contains a notable number of oxygen-containing groups, it provides remarkable opportunities for further modification with polymer either by ‘grafting to’ or by ‘grafting from’ techniques. (2) Non-covalent attachment such as electrostatic attraction, hydrogen bonding or π–π stacking; a variety of polymers have been incorporated into graphene-based films with a uniform multilayer structure and novel functionality, such as polyaniline [87–91], poly(sodium 4-styrenesulfonate) [92] and poly(p-phenylene vinylene) [48]. Recently, Mu et al. developed a series of PVDF-modified graphene monolayer papers with a gradient rGO/GO structure. In the gradient graphene paper, the GO region could respond to the environment stimuli including changes in humidity, temperature or light, leading to shrinking or swelling of the GO sheets [93]. In another example, poly-dopamine was used to functionalize graphene paper, the as-prepared graphene paper displayed high performance of responses to moisture, heat and light. The response of this water-driven actuator to multiple stimuli allows the artificial muscles and electric generators to be fabricated [94]. In our recent work, as shown in Figure 9, a highly branched PEI (b-PEI) was used for reduction and simultaneous derivation of GO to form a biocompatible polymeric matrix on rGO nanosheet. Ferrocene redox moieties were then grafted onto rGO nanosheets through the polymer matrix. The as-prepared functional composite is electrochemically active and enabled to accommodate enzymes stably. For proof-of-the concept studies, two crucial redox enzymes for biosensors (i.e. cholesterol oxidase and glucose oxidase (GOx)) were used to test the platform with good outcomes [95].
5. Applications of graphene paper as electrochemical-sensing platforms
Graphene papers with the advantages of low price, high quality and simple synthesis process have a potential to apply in electronic and optoelectronic devices, electrochemical energy devices, water treatment and sensors [7, 8, 10, 50, 51]. Especially the self-assembled 2D graphene papers/films/membranes functionalized with nanostructured metals, polymers and biomolecules are promising candidates for sensing applications, as summarized in Table 1, which is an overview of enzyme-based and non-enzymatic graphene papers/films sensors for the detection of glucose. In particular, in the field of electrochemical biosensors with high sensitivity, these candidates have found widespread uses in clinic diagnosis, environment monitoring and for quality control in industrial, food and agricultural products [9, 34, 59, 61, 96]. In Section 5, we mainly address the applications of graphene papers as different types of electrochemical sensor.
5.1. Enzyme-graphene paper sensors
Graphene paper displayed outstanding physical and electrochemical properties, such as high conductivity, large surface area, abundant defect sites, superior electrocatalytic activity and fast electron transfer rate, hence various enzyme biosensors based on this attractive material have been fabricated recently [11, 95]. We have successfully fabricated graphene paper doped with chemically compatible PBNPs as a nanohybrid electrocatalyst [36].
The correlation between the electrocatalytic current and glucose concentration shows a linear relation up to about 6–8 mM with a sensitivity of 25 μA mM−1 cm−2 and the detection limit down to about 10 μM. Furthermore, our group had recently explored a facile way for the successful synthesis of redox active and bioengineering of rGO for the development of versatile biosensing platform [95]. We developed a simple way for the synthesis of ferrocene (Fc) functionalization of highly b-PEI-linked rGO as well as the development of biocompatible matrix for accommodation of different bio-recognition elements. Ferrocene redox moieties were attached to rGO nanosheets through PEI. The polymer acts as both a reducing agent and a molecular spacer for rGO. Fc-PEI-rGO is electrochemically active and can offer a biocompatible microenvironment for immobilization of cholesterol oxidase and glucose oxidase. The as-constructed electroactive matrix was further used for the development of integrated biosensing platforms for cholesterol and glucose sensing. As measured, the current-time (
5.2. Non-enzymatic sensors for detection of small biomolecules
Glucose oxidase-modified electrode is the most common class of amperometric biosensors for glucose detection because GOx enables catalytic oxidation of glucose with high sensitivity and selectivity. However, enzyme-modified electrodes have some disadvantages such as the instability of the electrode and unsatisfactory reproducibility, complicated enzyme immobilization procedure and high cost of enzymes. For addressing such challenging issues, non-enzymatic glucose biosensors based on graphene papers have been explored. As mentioned above, nanostructured MO displayed high performance for the fabrication of electrochemical sensors. The working principles of using MO as an electrochemical catalyst for the detection of glucose can be described by Eqs. (1) and (2). A couple of redox peaks are obtained in a basic solution, and MO(OH) can then oxidize glucose to glucolactone, leading to a remarkable increase in the anodic peak current. Graphene paper plays a key role as supporting electrode and speeding up electron transfer reaction. Several studies have reported that MO-functionalized graphene papers can sensitively detect glucose without any biological materials involved in the fabrication of sensors. For example, CuO, a p-type semiconductor with a narrow band gap of 1.2 eV, is very promising for the development of glucose sensors because of high specific surface area, good electrochemical activity and the possibility of promoting electron transfer reactions at a lower overpotential. For example, CuO nanocubes were deposited on a graphene film by the EPD process, under the optimized conditions, a linear range up to 4 mM with a sensitivity of 1360 µA mM−1 cm−2 at a positive potential (i.e. 0.55 V) was achieved [100]. In order to improve the performance of CuO-modified graphene paper as non-enzymatic sensor, CuO-functionalized S-doped graphene was successfully synthesized through a facile microwave-assisted approach. The CuO/S-doped graphene-based sensor exhibited a rapid response of 2s (Figure 11), a wide linear range of 0.1–10.5 mM, a high sensitivity of 1298.6 μA mM−1 cm−2 and a low detection limit of 80 nM. Moreover, this biosensing platform showed good anti-interference ability and reasonable stability (Figure 11) [101]. In another case, PtNPs/MnO2 nanowires/graphene paper used as a free-standing paper electrode for non-enzymatic detection of glucose, the sensor response is linear to the glucose concentrations in the range from 0.1 to 30.0 mM with a detection limit of 0.02 mM (
Ascorbic acid (AA), dopamine (DA), uric acid (UA) and other small biomolecules are among the key small biomolecules that affect human health. Therefore, electrochemical sensing of those small biomolecules has played a vital role in monitoring the health. Recently, building electrochemical-sensing platforms based on graphene-supported nanomaterials for the detection of those small biomolecules has been a hot topic, due to the fast electron transfer kinetics and superior electrocatalytic activity of graphene-based materials. Dopamine is one of the mostly studied small biomolecules using graphene-based biosensors. Determination of dopamine, ascorbic acid and uric acid concentration is crucial, because the concentration level of these molecules in human body is closely linked to the health status. Graphene film-based electrodes as electrochemical sensors have shown high performance towards the analysis of dopamine, ascorbic acid and uric acid [104–110]. For example, Xia and co-workers developed a multifunctional electrochemical sensor based on N-doped graphene (NG), which can be used to simultaneously determine AA, DA and UA [105].
N-doped graphene exhibited high performance for electrochemical sensing of AA, DA and UA due to its unique structure and physiochemical property. The electrochemical sensor had a wide linear response to AA, DA and UA [105]. Moreover, some important small molecules for food safety control also could be detected by using graphene paper-based electrodes. As an example, an Orange II sensor was fabricated based on PtNP-functionalized rGO film. This sensor was evaluated for the detection of Orange II in an acetate buffer. The results showed a wide linear range (1 × 10−8–6 ×10−7 M) and the detection limit could be down to 3.4 × 10−10 M (at
5.3. Electrochemiluminescence sensors
Electrochemiluminescence (ECL) is a kind of luminescence induced by electrochemical reactions normally at liquid/solid interfaces. ECL has proven to be a highly sensitive and selective method and is a very useful tool in analytical science. The ECL method combines the advantages of chemiluminescent analysis with the ease of reaction control by applying an electrode potential. Therefore, ECL-based sensors have recently been developed using graphene papers. For example, based on Ru(bpy)32+ and alcohol dehydrogenase-modified graphene/bovine serum albumin composite film, ethanol biosensor was developed [122]. In another case, Li et al. developed an ultrasensitive ECL sensor for the detection of DA. In this study, cationic polythiophene derivative, poly[3-(1,10-dimethyl-4-piperidinemethylene)thiophene-2,5-diyl chloride] (PTh-D), was used to improve stability, and NH2–graphene acted as electron transfer accelerator. Under the optimal experimental conditions, the ECL signal linearly decreased with the increase of DA concentration in the range of 0.1–50.0 mM with a detection limit of 0.04 mM (Figure 12). This simple prepared ECL sensor also exhibited high selectivity, good reproducibility and long-term stability. The proposed ECL sensor was also evaluated by detecting DA in real samples [123]. Xu et al. obtained a paper-based solid-state ECL sensor based on poly(sodium 4-styrenesulfonate)-functionalized graphene/Nafion composite film. Very impressively, the detection limit could be down to (
5.4. Others
Immunosensors are among the most important sensors for biomedical applications. Due to high flexibility and conductivity, the development of graphene paper-based electrochemical immunosensors is highly desirable [125]. Indeed, there are some striking examples in this regard. For example, a simple and label-free electrochemical impedimetric immunosensor for immunoglobulin G (IgG) based on chemically modified graphene was prepared by the Martin group [126]. In another example, an efficient, low cost and robust impedimetric immunosensor for the rapid and sensitive detection of
6. Concluding remarks and outlook
In summary, worldwide researchers have explored graphene paper-based sensors by exploiting their unique advantages including high sensitivity, conductivity and in situ sensing. The recent research advances suggest that graphene paper-based materials could play a significant role in developing flexible sensors and electronic devices due to their intriguing structural and functional features. However, the progresses are accompanied by new challenges. Among the possibly critical challenges, the biocompatibility, electronic conductivity and stretchable capability are likely mostly concerned. In this regard, the further development of graphene papers by incorporation of biocompatible and conducting polymers is particularly encouraged. Although there are these challenges to be overcome in order to meet practically capable applications in real world, we would like to believe that flexible and free-standing graphene papers should continue emerging as a widely useful material platform for developing electrochemical-sensing technologies in the near future.
Acknowledgments
This work was supported by DFF_FTP, the Danish Research Council for Technology and Product Science (to Q.C., Project No. 12-127447). M.Z. acknowledges the CSC PhD scholarship (No. 201306170047).
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