Rapid detection of low concentration of specific analytes in small sample volumes is critical in the early point-of-care diagnosis. While conventional optical based detection is extremely sensitive [1
], they are time consuming and require expensive and complex optical imaging devices, sophisticated image recognition software and fluorescence dye labels. Alternatively, electrochemical biosensors utilizing enzyme-modified electrodes—coined as “enzymatic electrochemical biosensors”—have received considerable attention, due to their high sensitivity and specificity. Enzymes are very effective and precise biocatalysts, which perform and regulate certain processes in living systems. The performance of the enzymatic electrochemical biosensors greatly depend on enzyme immobilization, which involves the interaction between the enzyme and the host surface, and thus the surface properties of both plays a significant role in defining their functionality [3
]. In this context, graphene remains an ideal candidate for hosts due to its large specific surface area and excellent electrical characteristics. Graphene is a single-atom-thick nanomaterial with sp2
bonded carbon atoms arranging as a honeycomb structure [5
]. The two-dimensional nature of graphene offers numerous remarkable properties such as high carrier mobility, large specific surface area, and excellent electrical conductivity [6
]. For instance, graphene has been exploited for label-free electrochemical sensing due to the high electron transfer rates [9
]. Due to the excellent electronic conductivity, graphene based composites have been utilized for improving the biosensor performance [13
]. Recently, the graphene field effect transistor (G-FETs) based biosensors are gaining momentum due to its extremely high carrier mobility and capacitance [14
], where graphene surface is interfaced with various biomolecules and cells. In all these sensors, surface modification is an indispensable step to interface the graphene with biomolecules such as antibodies, cells, enzymes, or single strand DNA probes that can selectively bind/interact to the target biomolecules in solution during biosensor operation.
A wide range of covalent (with covalent bond formation) and non-covalent (due to van der Waals forces only) functionalization chemistries have been investigated to selectively functionalize the graphene surfaces [16
]. The most common way to functionalize graphene is via vigorous chemical oxidation processes, creating sp3
hybridized bonds that would affect the unique electrical properties of graphene [19
]. Alternative direct covalent functionalization (without oxidation intermediates) methods were developed, however, the low reactivity of graphene basal surface, long reaction time, and low surface coverage limits their practical applications. Conversely, non-covalent routes (pi-stacking) for functionalizing graphene were reported widely in the literature. However, the electrical conductivity of the functionalized graphene has been observed to significantly decrease compared to pure graphene. Moreover, the surface area of the functionalized graphene prepared by covalent and non-covalent techniques decreases significantly due to the destructive chemical oxidation of flake graphite followed by sonication, functionalization, and chemical reduction. Thus, it is critical to develop a non-destructive functionalization route to modify graphene surfaces with engineered sites for binding.
Grafting thin conducting polymer layers have attracted a great deal of attention over the past decade. Electrodes modified with polymer layers can be achieved by different methods, however, electropolymerization is attractive in the preparation of ultra-thin polymer layers because of its experimental simplicity, room temperature utility, degree of dimensional specificity, and precision over the layer thickness via controlling the charge passed and/or the potential at which the deposition is performed. Additionally, the electrochemical potential applied can shift the Fermi level of graphene, increasing its reactivity as compared to direct attack of the covalent sp2
bonds with aggressive chemicals. Recently, polydiaminonaphthalene (pDAN), a conductive polymer grafted from aromatic diamines, has been reported to exhibit interesting electrochemical characteristics and compatibility to binding biomolecules and to shorten the distance between the electrode surface and enzyme active sites [21
]. Nevertheless, pristine pDAN films exhibit low conductivity at neutral pH, which strongly affects the sensitivity of biosensors. Composites of pDAN with various other materials including carbon nanotubes, gold, Fe, etc., were reported to enhance the performance of the biosensor [21
]. Recently, Ngyuyen et al. explored the potential application of graphene-pDAN composite for biosensing application [26
]. It was reported that the current intensity of Pt electrodes modified with CVD graphene/pDAN is 20 times higher than Pt electrodes modified with pDAN. However, the mechanism of charge transport for pDAN on graphene is a key factor controlling the electrochemical sensing remains unexplored.
Furthermore, quantification of -NH2
groups is critical as it reflects the available active binding sites for the biomolecular immobilization. While a wide range of surface functionalization methods have been reported in literature, to the best of our knowledge, there are only very few articles report on the estimation of amine surface coverage. In 2011, Noel et al. reported the estimation of amino surface densities using calorimetric methods [27
]. Later, spectrophotometric based methods involving the intermediate dye formation for the quantification of amine groups onto ceramics [28
] and silica surfaces [29
]. Shiota et al. has demonstrated the quantification of surface amine moieties on inorganic and/or organic surfaces using cleavable fluorescent compounds [30
]. While the method offers good sensitivity, these techniques involve multiple steps. Thus, in this article, we report a controlled deposition of ultrathin functional polymer layers (1,5-diaminonaphthalene) onto CVD graphene surfaces using an electropolymerization technique and evaluated their surface characteristics. The influence of the polymer layer thickness on both the electrochemical properties as well as the enzyme immobilization was evaluated. A simple electrochemical method was demonstrated as a tool for quantifying the surface bound amine groups. Furthermore, the implications of such improved electrochemical performance and/or enzyme immobilization characteristics of ultrathin polymer layer grafted graphene towards enhancing the electrochemical biosensor performance was demonstrated using the classic horseradish peroxidase enzyme, as an example.
2. Materials and Methods
2.1. Materials and Reagents
Monolayer chemical vapor deposition (CVD) grown graphene was purchased from Graphenea and the samples were cleaved with 4 × 4 mm dimension. Sulfuric acid (ACS reagent, 95.0–98.0%, Sigma-Aldrich, Dorset, UK), 1,5-diaminonaphthalene (DAN) (99%, Aldrich), ferrocene carboxylic acid (97%, Sigma-Aldrich), phosphate buffered saline tablets (Sigma-Aldrich), horseradish peroxidase enzyme (Sigma), N-Hydroxysuccinimide (NHS) (98%, Sigma-Aldrich), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (>99%, Sigma-Aldrich) and H2O2 (99.5%, Sigma) were used as received.
The polymer films were deposited from 10 mM DAN in 0.25 M H2SO4 in a three-electrode system, where, monolayer CVD graphene was used as the working electrode, Pt wire as counter electrode and Ag/AgCl served as the reference electrode. The electropolymerization of monomer DAN produces polymer DAN (pDAN) on graphene surface generating NH2 groups on the surface. The thicknesses of the pDAN layers were controlled by controlling the number of cycles during the electropolymerization process.
2.3. Surface Functionalization
The surface coverage of amines on pDAN modified CVD graphene was estimated by functionalizing the amine groups with electroactive ferrocene carboxylic acid moieties. Typically, 250 µM ferrocene carboxylic acid was prepared in fresh PBS solution containing 5 mM EDC and NHS, which was incubated at room temperature for 40 min to activate the carboxylic functional group of the redox molecule. The resultant solution was drop-casted on a freshly prepared amine terminated pDAN modified CVD graphene surface and incubated at room temperature for 10 min. The electrode functionalization was performed prior to the electrochemical experiments and was used immediately.
2.4. Enzyme Immobilization
Aqueous solution of 5% glutaraldehyde was drop-casted onto the pDAN modified electrodes and was incubated for 30 min. Later, the electrodes were rinsed with de-ionized water (DI-water) and dried under nitrogen. The enzyme immobilization was performed by directly drop-casting 10 µL of horseradish peroxidase (HRP) enzyme (1 mg/mL) onto the modified surface and incubated at 4 °C for 18 h. The electrodes were rinsed with DI-water thrice to remove any weakly bound enzymes. The electrodes were freshly prepared and used immediately following the enzyme immobilization.
2.5. Characterization Techniques
The surface topography of pDAN modified graphene electrode was investigated using atomic force microscopy (AFM), (Dimension-3100 Multimode, Bruker, Billerica, MA, USA), and the AFM tip was a silicon-SPM sensor (contact mode), thickness 4 μm, length 125 μm, and width 30 μm. The chemical environment was studied using X-ray photoelectron spectroscopy (XPS). Electrochemical analysis was performed using the advanced potentiostat (PGSTAT-302 from Autolab, Metrohm Autolab, Runcorn, UK) with the scanning voltage in the range of −0.8 to 0.6 V for evaluating the electrochemical performance of enzyme-functionalized electrodes. Standard 3-electrode system was used for the electrochemical evaluation, where CVD graphene was used as the working electrode, Ag/AgCl as the reference electrode and Pt wire as the counter electrode. 0.01 M phosphate buffer saline (PBS) solution (pH = 7.4) was used as the electrode unless otherwise stated.