Electrochemical biosensor with graphene oxide nanoparticles and polypyrrole interface for the detection of bilirubin

Abstract

A bilirubin biosensor was fabricated by immobilization of bilirubin oxidase (BOx) on a graphene oxide nanoparticle (GONP) decorated polypyrrole (Ppy) layer electrochemically deposited onto a fluorine doped tin oxide (FTO) glass plate. The enzyme electrode (BOx/GONP@Ppy/FTO), Ag/AgCl as the standard electrode and platinum as the auxiliary electrode were assembled using a potentiostat to develop an amperometric bilirubin biosensor. The characterization of the enzyme electrode was fulfilled using Raman spectroscopy, scanning electron microscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The biological sensor demonstrated a response at pH 7.5 and 30 °C in only 2 s which was optimum once polarized at +0.2 V vs. Ag/AgCl. The biosensor’s limit of detection (LOD) and required limit of quantification (LOQ) was calculated as 0.1 nM (S/N = 3) and 2.6 nM, respectively. The electrocatalytic reaction illustrated a linear response over the bilirubin/substrate concentration in the range of 0.01 to 500 μM. The half-life of the biosensor is 150 days, during which it could be reused 100 times after keeping the biosensor at 4 °C. The bilirubin levels measured in serum samples of both healthy and jaundice afflicted persons correlated well with the popular colorimetric method, the coefficient of determination (R²) being 0.998.

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1. Introduction

Bilirubin is a product after the breakdown of heme by catabolism. Bilirubin is mainly excreted into bile as bilirubin glucuronides by hepatocytes.^1,2 Higher than normal levels (5–32 μM) of serum bilirubin may indicate liver dysfunction, damage in the brain, an inherited blood disorder – thalassemia, a genetic liver disorder Gilbert–Meulengracht syndrome, auto-hemolytic anemia, hemolytic uremic syndrome caused by hemolytic anemia, or sickle cell anemia.³ Although direct spectroscopic measurement⁴ and the diazo reaction⁵ are the two most commonly used methods for bilirubin determination, they have certain drawbacks. These drawbacks are the interference caused by other proteins present in the heme during direct spectroscopic measurement and pH dependency⁶ during the diazo reaction. In addition, there are certain analytical methods for measuring bilirubin including polarography,⁷ enzymatic assay,⁸ fluorimetric method,⁹ capillary electrophoresis,¹⁰ high-performance liquid chromatographic (HPLC),¹¹ chemiluminescence¹² and piezoelectricity,¹³ which require bulky and expensive instrumentation, long bilirubin test preparation time and a requirement for trained staff to carry out the procedure. Considering the above cons, it is not feasible for regular experimentation and detection. On the other hand, bilirubin oxidase (BOx) based biosensors are simple, as no sample pre-treatment is required, fast and more sensitive. These biosensors are based on either the consumption of oxygen¹⁴ or oxidation of hydrogen peroxide¹⁵ or using a complex formed as conductive poly-terthiophene–Mn(II)¹⁶ and a hybrid film which is fabricated by zirconia coated silica nanoparticles/chitosan.¹⁷ This will incur an electron transfer through Mn(II). However, the biosensors perform poorly, having a high detection limit and low stability due to inefficient electrical wiring of BOx with the electrode surface. Although some conducting materials such as poly-terthiophene and silica nanoparticles/chitosan hybrids have been employed to improve electron flow in these biosensors, results have been far from satisfactory.

The use of nanomaterials to improve biosensor performance has been quite popular since the last decade. Amongst the nanomaterials, graphene and its derivatives stand out from other nanomaterials owing to their potentially very high thermal and electrical conductivities and relatively low manufacturing costs.^18,19 They have a very large surface area although van der Waals interactions among each sheet of graphene cause aggregation, which effectively reduces their surface area. To solve this problem, hybrid materials of graphene with other nanomaterials are often synthesized.^20–22 Graphene forms sp²-bonded carbon atoms with six-atom rings and a reactive oxygen abundant group graphene oxide (GO) makes graphene’s surface very active resulting in association of additional and functional groups together.^23,24 The rapid electron transfer takes place at the surface of edge planes and defects when compared to the basal planes for the electrochemical sensors fabricated with graphene based materials.^25,26 The presence of these structural defects in chemically modified graphene can be exploited for electrochemical sensor applications.^27–30 Polypyrrole is the most commonly used conducting polymer in biosensors for their inherent stability in atmospheric conditions and biocompatibility.³¹ Polypyrrole (Ppy) presents a large surface area for immobilizing nanoparticles and enzymes or both due to its highly porous structure. Moreover, good electrical conductivity and stability of Ppy makes it an ideal material for biosensor development.^32,33 In order to avoid aggregation, GO was incorporated into polypyrrole (PPy). A strong chemisorption between carbon materials and polymers occurred.

A composite of GONP with Ppy (GONP@Ppy) is expected to behave better than either of the two materials used individually to enhance the biosensor response in terms of sensitivity, stability and better electrical connectivity. In our work, we firmly develop an innovative strategy for immobilizing bilirubin oxidase (BOx) on a GONP@Ppy modified fluorine-tin-oxide coated glass plate (FTO) electrode. Furthermore, characterization and optimization of the formed electrode and its application for amperometric determination of bilirubin in sera has been described in detail.

2. Experimental methods

2.1. Chemicals and reagents

BOx, 15 IU mg⁻¹ (EC 1.4.3.14 from Myrothecium verrucaria), 4-aminophenazone, Tris HCl, glutaraldehyde (GA) and FTO (100 mm × 100 mm × 2.3 mm) with a typical resistance of approximately ∼7 Ω sq.⁻¹ were bought from Sigma-Aldrich, St. Louis, USA. Bilirubin, phenol, horseradish peroxidase, BSA, NaClO₄, NaNO₃, K₃Fe(CN)₆, K₄Fe(CN)₆ and pyrrole from Sisco Research Laboratory, Mumbai, India and graphite rods from HB pencils from the local market were purchased. In all our experiments only deionized water (DW) was used. All other chemicals used were of analytical reagent grade.

2.2. Instruments

Electrochemical measurements were performed by Autolab PGSTAT 30 electrochemical workstation (Eco Chemie BV Utrecht, from Europe) having GPES 4.9 software. In the experiments, a modified FTO coated glass plate was used as the working electrode, KCl-saturated Ag/AgCl electrode as the reference electrode and a platinum electrode as the counter electrodes. Potential measurements were carried out with respect to the Ag/AgCl electrode and performed at 25–30 °C. The morphological characterization of GONP was performed by a high resolution transmission electron microscope at AIRF, JNU, N. Delhi. Scanning Electron Microscopy (SEM; ZEISS EVO® HD) experiments were carried out at Amity Institute of Advanced Research and Studies (AIARS), Amity University, Noida, India. 180° backscattering geometry was used to record Raman spectra. The excitation source was 532 nm from a diode pumped frequency doubled Nd:YAG solid state laser (Photop Suwtech Inc., GDLM-5015 L). The absorbance was taken using a UV-vis spectrophotometer (PerkinElmer, Lambda 750).

2.3. Assay of free BOx

The assay relies on measuring H₂O₂, released as a result of oxidation of bilirubin by BOx.^34,35 In the reaction mixture, 0.7 mL of Tris HCl buffer pH 8.5 (0.2 M), 0.1 mL of bilirubin solution (34.21 μM) and 0.1 mL of BOx solution (5 U mL⁻¹) were mixed at 37 °C for 10 min. After that, 1.0 mL of sodium phosphate buffer (0.4 M, pH 7.0), consisting of 50 μg 4-aminophenazone, 1.0 × 10³ μg solid phenol and 10 μg horseradish peroxidase was added to the reaction mixture & incubated in the dark at 37 °C for 15 min to develop the color, A₅₂₀ was read and H₂O₂ concentration was extrapolated from its standard curve.

The quantity of enzyme required for catalyzing the formation of 1.0 nmol of H₂O₂ by oxidizing bilirubin per min per mL under standard assay conditions is defined as one unit of enzyme.

2.4. Preparation of GONP

A graphite rod from an HB pencil was crushed to a powder in a pestle–mortar. Thus formed graphite powder (0.5 g) was initially dispersed in 23 mL of H₂SO₄ at 4 °C, and 0.5 g of NaNO₃ followed by 10 mL of KMnO₄ (2 mM) which was then added dropwise. The well-mixed slurry was stirred in a water bath for 1 h at 35 °C. Subsequently, 140 mL of DW was added in the mixture, and the temperature was raised to 90 °C for 15 min. After that, 3.0 mL of hydrogen peroxide (30 wt%) was injected causing the mixture to be converted into a fair brown colour. Consequently, GONPs were obtained by filtering, washing with DW and centrifugation at 4000 rpm.³⁶ TEM was used to characterized the finally obtained GONPs.

2.5. Electrodeposition of GONP@Ppy onto a FTO coated glass plate

GONP@Ppy was electrodeposited onto a FTO plate (100 mm × 100 mm × 2.3 mm) through cyclic voltammetry (CV). The FTO plate was immersed in a solution containing 50 mg of GONPs dissolved in 100 mM NaClO₄ (10 mL) and 30 mM pyrrole (4 mL) and a potential in the range of −0.2 to +0.8 V was applied up to 20 polymerization cycles with a scanning rate of 100 mV s⁻¹. The number of cycles influences the formation of the nanocomposite film onto the electrode. The thickness of electrochemically synthesized PPy films plays an important role in the response current of the biosensor. In previous reports, it has been observed that the response current is affected by the film thickness of nanocomposites. In general, thick films show a low response time, high sensitivity and a wider linear response range. It has also been suggested that the amount of enzyme entrapped in the film gradually increases with the increase of the polymerization cycle number.³⁷

The pyrrole solution from Sisco Research Laboratory, Mumbai, India was distilled prior to every deposition, and deaerated for 30 minutes using nitrogen gas. In the control, pure pyrrole was polymerized by the same procedure except no GONPs were mixed in the polymerization electrolyte. The electrochemical co-deposition of GONP@Ppy was studied by SEM, Raman spectroscopy and CV.

2.6. Preparation of enzyme electrode (BOx/GONP@Ppy/FTO electrode)

To prepare the enzyme electrode, a glutaraldehyde (GA) cross linked mixture for BOx and BSA was immobilized on the surface of the GONP@Ppy/FTO electrode. In order to make 30 μL of immobilization mixture, 10 μL of GA (2.5% v/v diluted in water) was added to 20 μL of phosphate buffer (0.1 M, pH 7.0) with 0.8 mg of BSA and 0.2 mg (3.0 IU) of BOx. The prepared crosslinked mixture of BOx and BSA (10.0 μL) was dropped carefully over the surface of the GONP@Ppy/FTO electrode and dried at room temperature. After 24 h, the electrode was thoroughly rinsed with five aliquots of 1.0 mL of phosphate buffer (0.1 M, pH 7.0), each for removing the residuals and unbound enzyme. Then the protein content of the washed out buffer was determined.³⁸

2.7. Response measurement of the BOx/GONP@Ppy/FTO electrode

CV measurements were performed using a 3 electrode cell with 20 mL of KCl as the electrolyte (0.1 M), 5 mL of sodium phosphate buffer (0.1 M, pH 7.5) and 0.1 mL of bilirubin (0.1 mM). The current (μA) was recorded through CV considering a potential range in between −0.4 and +0.4 V (vs. Ag/AgCl).

2.8. Optimization of the bilirubin biosensor

To measure the optimum enzyme concentration, the reaction was carried out with 100, 150, 250, and 500 IU enzyme concentrations. In order to optimize the biosensor performance, the pH effect, temperature, time of incubation and concentration of bilirubin on the immobilized BOx/GONP@Ppy/FTO electrode were studied. The optimum pH was determined at various pH ranges which were kept in between pH 7.0 to 10.0 at intervals of 0.5. The buffers each had a final concentration of 0.1 M: pH 7.0 to 7.5 for sodium phosphate buffers and pH 8.0 to 10.0 for Tris HCl buffer were used. Similarly, in order to determine the optimum temperature, the reaction mixtures were kept at various temperatures (20–50 °C) in increments of 5 °C and time (1–10 s) at an interval of 1 s. The concentrations of bilirubin in the range of 0.01–500 μM were considered for observing the fabricated biosensor response on various bilirubin concentrations.

2.9. Application of the bilirubin biosensor in sera

Blood samples from healthy volunteers (male/female) and jaundice patients (male/female) were obtained with their previously written informed consent from the Pt. BDS University of Health Sciences, Rohtak (Haryana), India. The blood samples were kept for 20 min at room temperature and then centrifuged at 1000–2000 × g for 10 min to remove the clot. The resulting supernatant was designated as serum, which was kept at 2–8 °C in a clean polypropylene tube for immediate use. These serum samples were stored at −20 °C into 0.5 mL aliquots, when not analyzed immediately. Total bilirubin in serum was determined by the above method except the bilirubin solution was used instead of serum and testing was carried out under optimal assay conditions. The concentration of bilirubin in serum was evaluated by a standard curve between bilirubin concentration vs. current in microampere prepared with the optimal assay conditions of the enzyme/GONP/Ppy modified FTO electrode. The experiments had been approved by the University ethics committee.

2.10. Storage stability of BOx

Storage stability of the enzyme modified electrode at 4 °C in dry conditions was tested for 5 months, where the enzyme was assayed intermittently on every 7^th day for 5 samples.

3. Results and discussion

3.1. Characterization of GONPs and the modified electrode

The GONPs were characterized by recording their X-ray diffraction (XRD) pattern with an X-ray diffractometer (Make: 122 Rigaku, D/Max2550, Tokyo, Japan) and transmission electron micrograph with a TEM, (Fig. 1A and B). Fig. 1A(a) shows XRD analysis through Hummer’s method which is modified for the synthesis of GONPs. A diffraction peak for the GONP was observed at 2θ = 12.02° having 0.77 nm interlayer distance.³⁹ The stacking height for the synthesized GONP calculated by the Scherrer equation was 9.1 nm. The number of layers ordered through d-spacing was ∼12. The absence of characteristic peaks for impurities revealed the high purity of the GONPs. XRD patterns of Fig. 1B(b) depicted that the Ppy/GO composite nanosheets are essentially amorphous. A broad peak appears at 2θ = 22.4° signifying an amorphous Ppy and GO. Fig. 1B shows a HRTEM image of GO sheets revealing the lattice fringes of graphene. Additional information was given about the interplanar distance d for GO material (3.610 Å).

Fig. 1 (A) X-ray pattern of (a) GONPs and (b) Ppy/GONPs. (B) HRTEM image of GONPs.

A smooth surface of the bare FTO electrode was visualized (Fig. 2a). After homogeneous dispersion of GONPs in the Ppy network, a uniform granular porous morphology of the GONP@Ppy/FTO electrode appeared (Fig. 2b). The SEM image of the surface of the BOx/GONP@Ppy/FTO electrode is shown in Fig. 2c. After BOx immobilization, the uniformly dispersed globular structure appeared due to the interaction between the BOx enzyme and the GONP@Ppy/FTO electrode.

Fig. 2 Scanning electron microscopy (SEM) images of (a) the bare FTO electrode, (b) GONP@Ppy/FTO electrode and (c) BOx/GONP@Ppy/FTO electrode.

3.2. Surface morphological studies

AFM was performed to investigate the morphological characteristics of the Ppy/FTO and GONP@Ppy/FTO composite film (Fig. 3). A magnified image of Ppy/FTO (3D visualization in inset) is shown in Fig. 3a. The average roughness of the Ppy film has been found to be 1.3 nm. After electrochemical oxidation of the GONPs, changes in the morphology of the film were observed. The GONP@Ppy/FTO magnified image and 3D image are shown in the inset of Fig. 3b, respectively. GONP/Ppy was uniformly spread on the glass substrate which is shown in the micrograph. As deposition of the GONPs occurred uniformly, this resulted in size homogeneity making a well structured film formation. The increase in the roughness of the film (2.3 nm) confirms modification of GONPs by the Ppy film. The increased thickness after immobilization of the enzyme on the film was measured as 3.1 nm (c).

Fig. 3 (a) AFM images of the Ppy/FTO electrode with an inset zoomed image of the Ppy film, (b) 3D image of the GONP/Ppy/FTO electrode and (c) 3D image of the BOx/GONP@Ppy/FTO electrode.

3.3. FT-IR study of the GONPs, Ppy and GONP/Ppy nanocomposite

GONPs, Ppy and the GONP/Ppy composite were characterized by a FT-IR study. The spectrum of the GONPs shows the respective peaks at 1055 and 1730 cm⁻¹ for C–O (alkoxy) stretching and C=O group stretching vibrations. Peaks at 3400 and 1413 cm⁻¹ are ascribed to the vibration and deformation of the O–H group.⁴⁰ The characteristic peak at 1620 cm⁻¹ corresponds to the vibration associated with remaining graphitic C=C.⁴¹ Peculiar peaks at 1546, 1457 and 3445 cm⁻¹ in the FTIR spectra of Ppy are due to the pyrrole ring C–C, C–N and N–H stretching vibrations respectively. In addition, the peaks at 1305 for C–N stretching vibrations, at 1698 cm⁻¹ for C=O stretching vibration and at 1038 cm⁻¹ for C–H deformation vibrations are also present³³ (results not shown).

FTIR spectra of the GONP/Ppy composite show every characteristic peak of the GONPs and Ppy except for slight changes. In regards to the peak of C=O stretching vibrations associated with GONPs, it shifted from 1730 to 1680 cm⁻¹, which is probably due to the π–π interactions and hydrogen bonding between GONP sheets and aromatic Ppy rings.⁴¹ For the pyrrole ring, the peak corresponding with C–C stretching also shifted slightly from 1546 to 1537 cm⁻¹, which could be assigned to π–π interactions between the unoxidized domain of the GONPs and pyrrole rings of Ppy. The results confirmed that GONPs and Ppy were not just physically present together but also interacted chemically via formation of π–π bonds.

3.4. Construction of the bilirubin biosensor

Fig. 4 shows the fabrication of the enzyme electrode based on immobilization of BOx on the GONP@Ppy modified FTO electrode. A mixture of Ppy and GONP was co-deposited through an electrochemical approach on the bare FTO electrode, as the method is simple and it is easy to control the thickness of the deposited layer. Further modification of the GONP@Ppy/FTO electrode with the enzyme was done by adsorption of the extensively crosslinked solution of BOx–BSA–glutaraldehyde over the electrode surface. Out of two –CHO groups of glutaraldehyde, the one –CHO group was attached to the –NH₂ group on the surface of the enzyme and another –CHO group was linked to the –NH₂ group of BSA. This cross linking forms a stable complex of BOx. The CV of GONP@Ppy/FTO shows elevated currents compared to Ppy/FTO indicating a high potent surface area and enormous conductivity of the GONP@Ppy/FTO composite film compared to the Ppy/FTO composite film. Overall, it can be safely resolved that the presence of GONPs provides faster charge transfer kinetics and thus enhances the biosensor response and increases its sensitivity.

Fig. 4 Schematic illustration of the stepwise amperometric bilirubin biosensor fabrication process.

3.5. Electrochemical impedance spectroscopy (EIS) and Raman spectroscopy

Electrochemical impedance spectroscopy (EIS) is an analytical tool to study the change in impedance of the electrode surface, a comparative account of which at successive stages of electrode fabrication confirms the successful modification of the electrode. The linear region under the lower frequencies indicates a Warburg diffusion in contrast to the Nyquist plot of the semicircle part at higher frequencies which is equivalent to the charge transfer resistance (RCT).⁴² The Nyquist plot (Fig. 5A) was constructed with the respective electrodes in 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM K₃Fe(CN)₆/K₄Fe(CN)₆ (1 : 1) as a redox probe. The RCT values for the bare FTO (curve a), GONP@Ppy/FTO (curve b) and BOx/GONP@Ppy/FTO (curve c) electrode were obtained as 750 Ω, 310 Ω and 520 Ω, respectively. The RCT of the GONP@Ppy/FTO electrode was lower than the FTO electrode, revealing its enhanced conductivity, lower resistance and the efficiency of the electron transfer which is on the higher side. The GONP immobilized onto Ppy were layered as a thin GO film. This suggests that a composite of GONP@Ppy has more active sites for faradaic reactions and a larger capacitance than the FTO electrode. The RCT of the BOx/GONP@Ppy/FTO bioelectrode is demonstrated by an enlargement of the semicircular diameter after the immobilization of BOx on GONP@Ppy employing a higher thickness, hydrophobic material property and compactness causing a resistance to electron transfer. Moreover, these results showed the immobilization of BOx onto the GONP@Ppy composite.

Fig. 5 (A) Nyquist plot obtained for the bare FTO electrode (a), GONP@Ppy/FTO electrode (b) and BOx/GONP@Ppy/FTO electrode (c) in 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM K₃Fe(CN)₆/K₄Fe(CN)₆ (1 : 1). (B) Raman spectra obtained for the (a) GONPs, (b) Ppy and (c) GONP@Ppy composite.

Characterizations of the GONPs, Ppy and GONP@Ppy composite were performed by recording their Raman spectra. The ratio of intensity of the D/G band (I_D/I_G) is a measure of defects present in the graphene structure, where the D band represents out of plane bending vibrations of sp³ bonded carbon atoms and the G band stands for in plane bending vibrations of sp² bonded carbon atoms in the structure.⁴³ As evident from Fig. 5B, characteristic peaks for the D band around 1357 cm⁻¹ and for the G-band at 1595 cm⁻¹ were present and the calculated I_D/I_G for GONPs was 0.99. For pure Ppy, characteristic stretching vibrations of the ring and C=C backbone were observed at 1380 cm⁻¹ and 1577 cm⁻¹ respectively.⁴⁴ In addition, the peak at 928 cm⁻¹ and two small peaks at 964 cm⁻¹ and 1060 cm⁻¹ are due to the bipolaron ring deformation and the polaron symmetric C–H in-plane bending vibration, respectively.^45,46 The calculated I_D/I_G ratio for Ppy is 0.88. The Raman spectrum of the reduced GONP@Ppy composite showed the bands for pyrrole, and proved the presence of Ppy in the composite. Additionally, the decline of the I_D/I_G value (0.82) of GONP@Ppy established the presence of less disordered graphene in the composite. The reduction of GO through the electrodeposition process is confirmed by the peak shift of the G-band from 1590 cm⁻¹ to 1570 cm⁻¹ compared to GONP.⁴⁷

3.6. Electrochemical study of the GONP@Ppy composite modified electrode

Fig. 6A represents the cyclic voltammograms recorded for a (a) GONP modified FTO electrode and (b) Ppy modified FTO electrode. The obtained values on the GONP/FTO electrode and Ppy modified FTO electrode were 0.13 mA and 0.15 mA, respectively. When the electrode was modified by GONPs, the current response of CV was increased [curve (a)]. The electrodeposition of GONPs on the FTO electrode surface led to a vast increase in current intensity as a result of the increase in the electro-active area. Sequential fabrication of the enzyme electrode as studied by CV at a potential of +0.2 V, is depicted in Fig. 6B. As expected, the bare FTO electrode (curve a) did not show any definite redox peak and the current characteristic of Ppy (Ppy/FTO) was spotted after 10 potential scans (curve b). CV of GONP/Ppy/FTO in curve c showed an elevated level of current with well characterized oxidation and reduction peaks, credited solely to the presence of GONP. Strong adsorption ability and high catalytic efficiency of GONPs also makes electrochemical co-deposition of GONP@Ppy composite layers comparatively more feasible than the pure Ppy layers. At +0.15 mA (ox.), the BOx/GONP/Ppy/FTO showed a redox peak corresponding to the oxidation of H₂O₂ by immobilized BOx (curve d).

Fig. 6 (A) Cyclic voltammograms recorded for a (a) GONP modified FTO electrode and (b) Ppy modified FTO electrode. (B) Cyclic voltammograms recorded for a (a) bare FTO electrode, (b) Ppy/FTO electrode, (c) GONP@Ppy/FTO electrode and (d) BOx/GONP@Ppy/FTO electrode in a sodium phosphate buffer solution (pH 7.5) containing 0.1 mM bilirubin.

3.7. Response measurements of the bilirubin biosensor

The optimum amount of enzyme for the preparation of the working electrode was 150 IU (20 μL). The enhancement of the oxidation current by increasing bilirubin concentration is achieved by the increased concentration of H₂O₂ during enzymatic reaction. The amperometric response studies of the BOx/GONP/Ppy/FTO electrode in bilirubin concentrations from 0.01–500 μM using phosphate buffer (0.1 M, pH 7.5) are shown in Fig. 7A.

Fig. 7 (A) Effect of substrate concentrations (a) without substrate, (b) 0.01, (c) 1.0, (d) 100, (e) 200, (f) 300, (g) 400 and (h) 500 μM on the response of the BOx/GONP@Ppy/FTO electrode in 0.1 M sodium phosphate buffer solution (pH 7.5) containing 5 mM K₃Fe(CN)₆/K₄Fe(CN)₆ (1 : 1) as a redox probe at a potential range of −0.4 to ∓0.4 V and a scan rate of 50 mV s⁻¹ vs. Ag/AgCl reference electrode. Inset: the calibration curve between the bilirubin concentration (μM) and current response (mA) of the biosensor. (B) Chronoamperometric curve measured in the presence of different concentrations of bilirubin.

A linear relationship was found between the current (in μA) and the bilirubin concentrations in the range 0.01–500 μM (inset of Fig. 7A), which is wider than reported earlier.^16,17,48,49 Current–time plots for various concentrations of bilirubin are shown in Fig. 7B. Voltammetric measurements were performed after each addition of bilirubin to a maximum concentration of 500 μM. No significant increase in the current was observed when the concentration of bilirubin was increased beyond 500 μM, indicating that the electrode had reached its saturation level at this bilirubin concentration. The time required to attain 95% of the steady-state response was within 2 s, which indicates a very fast dilution process.

The limit of detection (LOD) and quantification (LOQ) of the biosensor were calculated as 0.1 nM (S/N = 3) and 2.6 nM, respectively. This is considered a better biosensor than previously constructed sensors since the modification of the quartz crystal is carried out by using a hydroxyapatite (HAP) film through the process of molecular imprinting and the surface sol–gel technique (0.01 mM).⁴⁸ Furthermore, the application of gold nanoclusters as the fluorometric and colorimetric probe⁴⁹ and conductive poly-terthiophene–Mn(II) complex¹⁶ for the GONP/Ppy/FTO electrode provided a faster electronic conductance between the electrode and enzyme maintaining a remarkable biocompatibility.

3.8. Optimization of the biosensor

In order to investigate the maximum amount of GONPs that can be deposited along with Ppy, their concentration was varied from 0.1 to 10 mg mL⁻¹ in the NaClO₄ + pyrrole + GONP solution and the chronoamperometric current response was recorded. Though the anodic current increased with increasing concentration of GONPs, accompanied by changes in the morphology of the GONP/Ppy composite, substantial enhancements were not observed towards higher concentrations of GONPs (ESI Table S1†). Hence for preparing the working electrode, a 5.0 mg mL⁻¹ concentration was employed. Diaz’s mechanism (pyrrole electropolymerization⁵⁰) indicated that the oxidation of pyrrole monomers produces a solution rich in the pyrrole radical cation (Py*+) near the electrode surface. These unstable Py*+, may react with anions present in the solution to form low molecular weight soluble products or they may bond with GONPs, via hydrogen bonding and electrostatic interactions.⁵⁰ Since GONPs exhibit a high surface area to volume ratio, they therefore act as a specific site for soluble products having low molecular weights.

The voltage of +0.2 V was taken for all the experiments as standard since the optimum response of the biosensor was observed at this voltage. The effects of pH, temperature, incubation time and the bilirubin substrate concentration were evaluated as these factors affect the experimental conditions in response to the biosensor. At the temperature of 30 °C and pH of 7.5, the optimum current was recorded. The optimal pH value is lower than that for bilirubin monitoring based on the indirect electrochemical response (pH 8.5)¹⁰ and glassy carbon modified with ferrocenecarboxamide and AuNP and multiwalled carbon nanotubes (pH 8.0).⁵¹ A linear relationship was obtained between biosensor response and substrate (bilirubin) concentration from 0.01–500 μM; the response was constant after 500 μM. The novel biosensor exhibits a high sensitivity of 0.914 μA μM⁻¹. The biosensor responded very quickly, within 2 s it records 95% of the constant current at each point.

3.9. Evaluation of the biosensor

Reproducibility of the present sensor was evaluated by analyzing % recoveries of the added bilirubin. Serum samples spiked with 5.0 and 10.0 mM bilirubin, showed 96.1 and 97.5% recoveries. Precision and test–retest reliability of the assay were checked through relative standard deviation (RSD) values. Furthermore, the results obtained for detection of bilirubin in serum samples for intra-assay and inter-assay RSD values were 3.6% (same day) and 4.27% (after storage for a week). A remarkable consistency of the results were revealed by our method attributed to the pooled presence of GONPs and Ppy on the enzyme electrode.

3.10. Detection of bilirubin in real samples

The bilirubin levels were measured in the constructed biosensor and were found ranging from 0.1–16 and 21–61 μM in the sera samples of apparently healthy and jaundice afflicted persons respectively (ESI Table S2†). In order to evaluate the accuracy of this method, 20 serum samples were compared for bilirubin detection both by the BOx/GONP/Ppy/FTO electrode (y) and the popular colorimetric method (x). The experiment was done in triplicate. Correlation analysis using the regression equation showed a linear relationship, with a coefficient of determination (R²) of 0.998, and the regression equation as y = 1.0275x − 1.8665 (Fig. 8). These results highlighted the analytical performance of the current biosensor in biological (serum) samples showing a better response compared to other biosensors.

Fig. 8 Correlation between serum bilirubin values measured by the chemical photochlorometric method (x-axis) and the current method (y-axis) employing the bilirubin biosensor based on the BOx/GONP@Ppy/FTO electrode.

3.11. Interference study and selectivity

Interference from ascorbic acid (0.05 mM), glucose (5 mM), glycine (0.4 mM), uric acid (0.2 mM) and creatinine (0.1 mM) at their physiological concentration was investigated. The interferents were added for comparing the difference in amperometric response before and after addition. For all assays, the concentration of bilirubin (100 μM in 0.1 M sodium phosphate buffer, pH 7.5) was kept constant. The addition of the interference at the applied potential resulted in activity decreases of only 3% for ascorbic acid, 2.0% for glucose, 3% for glycine, 4% for uric acid and 1% for creatinine (Fig. 9A). Hence, these metabolites/interferences have practically no affect on the biosensor response.

Fig. 9 A) Effect of interferents on the activity of the BOx/GONP@Ppy modified ITO electrode with 100 μM bilirubin in 0.1 M sodium phosphate buffer (pH 7.5). (B) Effect of storage stability at 4 °C on the response of the bilirubin biosensor based on the BOx/GONP@Ppy modified FTO electrode. (C) Effect of storage at 4 °C on the response of three similar BOx/GONP@Ppy modified FTO electrodes.

3.12. Determination of frequency for reusability, uniformity and stability of the enzyme electrode over time

To determine whether our biosensor is stable over time, the current response of the biosensor was routinely measured while keeping it in intermittent storage (4 °C). For as many as 100 times, the enzyme electrode was reused during 150 testing days which consequently retained 50% of its initial activity (Fig. 9B), showing significant advancement over the earlier reported results.^{16,17,48,52,53} Triplicates of the enzyme electrode were constructed and evaluated individually for the storage effect at 4 °C. As evident from Fig. 9C, no significant difference in the storage stabilities of the prepared electrodes were observed indicating a remarkable and satisfactory performance and demonstrating high stability and productivity pertaining to the higher frequency for using the enzyme electrode.

Comparing with the previous analytical methods, for instance electrochemical and piezoelectric methods (ESI Table S3†), this amperometric enzyme sensor exhibited a higher sensitivity, lower detection limit and faster implementation for bilirubin detection. The linear range of this sensor for bilirubin is 0.01 to 500 μM. Consistencies of the results from our detection method compared with that of the standard method are observed. The novelty of our biosensor has showed its promising application in point-of-care testing (POCT) for rapid in vitro diagnosis of jaundice.

4. Conclusion

In our present work, we have successfully designed a signal amplified electrochemical enzyme sensor for bilirubin based on the synergistic catalysis of BOx and GONP@Ppy nanostructures. The use of a BOx/GONP@Ppy modified FTO electrode has facilitated an analytical upgrade of the bilirubin biosensor in terms of its limit of detection which is 0.1 nM, a vast working concentration range in between 0.01 and 500 μM, rapid response (within 2 s) and higher storage stability of 150 days without any intervention by many compounds compared with other biosensors. We found that the excellent electrocatalytic activity, biocompatibility and stability of GONP@Ppy would offer a new platform for biosensors.

Conflict of interest

Authors state that they have no competing interests.

Abbreviations

BOx	Bilirubin oxidase
GONP	Graphene oxide nanoparticles
Ppy	Polypyrrole
FTO	Fluorine doped tin oxide glass plate

Acknowledgements

The authors (Nidhi Chauhan, Rachna Rawal and Utkarsh Jain) gratefully acknowledge the financial support from Science & Engineering Research Board, Department of Science and Technology (SERB-DST), New Delhi.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15671a

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