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Glucose Biosensor Based on Silicon Nitride Nanoparticles Abdollah Salimi ,*a, b Roaya Zand-Karimi,a Abdollah Noorbakhash,a Saied Soltanianb a
Department of Chemistry, University of Kurdistan, P. O.Box 416, Sanandaj, Iran Research Center for Nanotechnology, University of , Kurdistan , P. O.Box 416, Sanandaj, Iran tel: + 98-871-6660075, fax: + 98-871-6624001 *e-mail:
[email protected];
[email protected] b
Received: February 17, 2010;& Accepted: May 24, 2010 Abstract For the first time silicon nitride (Si3N4) nanoparticles was used for preparation electrochemical biosensor. GOx immobilized on the Si3N4 nanoparticles exhibits facile and direct electrochemistry. The surface coverage and heterogeneous electron transfer rate constant (ks) of immobilized GOx were 6.3 1013 mol cm2 and 47.4 0.3 s1. The sensitivity, linear concentration range and detection limit of the biosensor for glucose detection were 38.57 mA mM1 cm2, 25 mM to 8 mM and 6.5 mM, respectively. This biosensor also exhibits good stability, reproducibility and long life time. These indicate Si3N4 nanoparticles is good candidate material for construction of third generation biosensor and bioelectronics devices. Keywords: Glucose oxidase, Silicon nitride, Nanoparticles, Direct voltammetry, Biosensors
DOI: 10.1002/elan.201000131
1. Introduction Electrical contacting of redox enzymes with electrode supports attracts substantial research efforts directed to development biofuel cells, bioelectronic devices and biosensors [1]. The combination of nanomaterials and biomolecules is of interest to fabrication biosensors due to interesting properties of nanomaterials such as, large surface to volume ratio, high surface reaction activity, high catalytic activity, strong adsorption ability and excellent biocompatibility [2, 3]. Furthermore, nanomaterials have a unique ability to promote fast electron transfer between the active site of the enzyme and electrode surface. Different nanomaterials such as gold nanoparticles [4], graphite nanosheet [5] carbon nanotubes (CNTs) [6, 7] metal oxide nanomaterials [8, 9], NdPO4 nanoparticles [10] CNTs-gold nanoparticles [11, 12], Pt nanoparticles-CNTs/CdS [13], quantum dots/CNTs [14], CNTs-ionic liquid (IL) [15] and SiO2 nanoparticles [16] have been widely used in constructing electrochemical glucose biosensors. Although direct electrochemistry was obtained for more modified electrodes, only few reversible electrochemical behavior of immobilized glucose oxidase was observed so far. Moreover, the catalytic activity of the immobilized enzyme was low. In addition the immobilization process have been long and required specific reagents. Since the enzymes undergo serious structure change during its immobilization on the surface of electrodes, and its bioactivity may be weakened or even destroyed in the immobilization procedure, it is still a great challenge to create novel surfaces that can fix enzymes firmly without alerting its original conformation and bioactivity. 2434
Silicon nitride (Si3N4) plays an important role in microelectronics, integrated circuits technology, memory and thin film transistors, optoelectronics, optics and hard surface coating [17–19]. The photoelectrochemical behavior of silicon single crystal electrode covered with a thin film of Si3N4 in aqueous electrolyte (natural pH) has been studied [20].The application of Si3N4 thick film for fabricating and developing of novel biosensor have been reported [21–23]. However, to the best of our knowledge, the application of silicon nitride nanoparticles for fabrication of voltammetric and amperometric biosensors has not been studied. Here we report, for the first time, the fabrication, characterization and analytical performance of a glucose biosensor based on incorporation of glucose oxidase enzyme onto Si3N4 nanoparticles at the surface of GC electrode. Direct electron transfer and bioelectrocatalytic activity of immobilized enzyme were investigated by cyclic voltammetry and amperometry techniques. The GC electrode modified with Si3N4 nanoparticles shows excellent biocompatibility toward GOx. The resulting electrode was applied as glucose biosensor in the absence and presence of electron transferring mediator (ferrocene methanol) for micromole detection of glucose.
2. Experimental 2.1. Chemical and Reagents Glucose oxidase (EC 1.1.3.4. Type II: from Aspergillus niger) was purchased from Sigma. 31.25 mM GOx solutions (pH 7) were stored at 4 8C. Silicon nitride nanoparticles with diameters 15 5 nm were obtained from
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MERCK. The glucose, phosphate buffer solutions (PBS), ferrocenemethanol (Fc) and other reagents used were of analytical reagent grade. Pure argon was passed through the solution to avoid possible oxygen action during the experiments.
2.2. Instrumentation Electrochemical experiments were performed with a computer controlled m-Autolab modular electrochemical system (Eco Chemie Ultecht, The Netherlands), driven with GPES software (Eco Chemie). A conventional three-electrode cell was used with a Ag/AgCl (sat. KCl) as reference electrode, a Pt wire as counter electrode and a GC disk (2 mm diameter) as working electrode. The effective surface area of the electrode modified with Si3N4 nanoparticles was determined as 0.07 cm2 from CV of 1 mM K3[Fe(CN)6].
2.3. Modification of GC with Si3N4 Nanoparticles The GC electrode (2 mm diameter) was carefully polished with alumina on polishing cloth, and then ultrasonicated in water bath for several minutes to remove adsorbed particles. Then 50 mL of EtOH-Si3N4 solution (0.5 mg/ mL) was cast on the surface of GC electrode.
2.4. Immobilization of GOx on Si3N4 Nanoparticles 10 mL of freshly GOx solution (31.25 mM, in pH 7) was spread onto the desired Si3N4/GC modified electrode. The bioelectrode was kept at room temperature for about 20 min. Finally this bioelectrode was washed by PBS to remove any unbound enzyme.
3. Results and Discussion 3.1. Direct Voltammetry of the Immobilized GOx onto Si3N4 Nanoparticles The morphology and particle size of Si3N4 powder was examined by TEM. As shown Si3N4 powder is very uniform and fine with average grain size of 15 5 nm (Figure 1). Cyclic voltammograms of GC/Si3N4 and GC/Si3N4/GOx modified electrodes in pH 7 PBS at a scan rate of 20 mV s1 was recorded. At potential range 0.25 to 0.65 V no redox peaks can be seen for GC electrode modified with silicon nitride nanoparticles. However, a pair of reduction–oxidation peaks with formal potential 0.44 V vs. reference electrode is clearly observed for GC/Si3N4/GOx modified electrode. It confirms that the redox peak pairs appeared at GOx/Si3N4 modified glassy carbon electrode is correspondent to the real direct redox behavior of the enzyme redox center (FAD). The separation of anodic to cathodic peak potentials (DEp) is 30 mV and ratio of anodic to cathodic peak currents is about one. This indicates that glucose oxidase undergoes a reversible redox process at the glassy carbon electrode modified with Si3N4 film. Thus, Si3N4 film has a great effect on the electrode kinetics and provides a suitable environment for the glucose oxidase to transfer electron with underlying GC electrode. These further suggest that the silicon nitride nanoparticles greatly facilitates active site of GOx to approach the electrode, therefore the electron transfer rate constant of immobilized GOx increased. The formal potential of GOx at Si3N4 nanoparticles (E8’) is close to the formal potential of glucose oxides immobilized at the surface of other nanomaterials modified electrodes[4–16]. Figure 2B shows the Nyquist plots of GC electrode (curve a), GC/Si3N4 electrode (curve b) and GC/Si3N4/
Fig. 1. TEM image of Si3N4 powder. Electroanalysis 2010, 22, No. 20, 2434 – 2442
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Fig. 2. (A) Cyclic voltammetric response of GC electrode modified with Si3N4 nanoparticles(a) and GC electrode modified with Si3N4 nanoparticles and GOx in PBS pH 7 and scan rate 20 mV s1 (B) Nyquist plots for GC electrode (a), GC electrode modified with Si3N4 nanoparticles (b), and GC electrode modified with Si3N4/GOx (c) in 0.1 M KCl solution containing 1 mM [Fe(CN)6]3/4.
GOx (curve c). The straight line at low frequency is related to the diffusion process known as Warburg element, while the high frequency semicircle is related to the electron transfer process which combining the charge transfer resistance and double layer capacitance and can be used to determine whether an electrochemical reaction proceeds with ease or difficulty according to the diameter of the semicircle [24]. As can be seen, after the immobilization of GOx onto GC/Si3N4 nanoparticles the value of Ret 2436
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is significantly increased to about 4500 W (curve c). The increased value of Ret obtained for GC/Si3N4/GOx indicates hindrance to the electron transfer, confirming the successful adsorption of GOx onto Si3N4 film. The depressed semicircle arc shows the GC electrode modified with Si3N4 nanoparticles has an ideally flat surface [25]. Therefore a uniform thin film of Si3N4 is immobilized onto GC surface.
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Fig. 3. (A) Cyclic voltammetric response of Si3N4/GOx/GC electrode at different scan rates , from inner to outer 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s1, ( B) Plot of peak currents vs. scan rates.
Cyclic voltammograms of GC/Si3N4/GOx modified electrode at different scan rates were recorded (Figure 3). Both the anodic and cathodic peaks currents are linearly proportional to the scan rate in the range of 10 to 2000 mV s1 (R2 = 0.999), as expected for surface confined redox process. The peak current is proportional to v , in contrast to the v1/2 dependence observed for nernstian waves of diffusing species[26, 27]. Moreover, redox potentials are almost the same at the different scan rate. It is clear that GOx adsorbed onto the surface undergoes a reversible electron transfer with the Si3N4 nanoparticles.
The peak to peak separation is about 35 mV at scan rates below 100 mV s1, suggesting facile charge transfer kinetics over this range of sweep rates. According to the Laviron theory [28], the transfer coefficient (a) and electron transfer rate constant ks of immobilized GOx at Si3N4 nanoparticles were about 0.49 and 47.4 0.3 s1, respectively. The observed electron transfer rate constant is higher than ks values of GOx at different nanomaterials modified electrode [4–16] (Table 1). High ks value implying that the Si3N4 nanoparticles may be more favorable for direct electron transfer of GOx. It is assumed that the
Table 1. Analytical parameters for glucose biosensors fabricated based on different nanomaterials. Electrode
Method
Dynamic range (mM)
Limit of detec- Sensitivity ks tion (mM) (mA/mM) (s1)
GCE [a] modified with TiO2 nanostructures [9] GCE modified with nanosheet [5] GCE modified with CNTs [b] [6] GCE modified with CNTs and quantum dots [14] GCE modified with NiOx nanoparticles [8] Nitrogen doped CNTs [7] Pyrolytic graphite electrode modified with SiO2 nanoparticles [16] GCE modified with NdPO4 anoparticles [10] GCE modified with gold nanoparticles [4] GCE modified with CNTs/gold nanoparticles/polymer film [12] GCE modified with IL/CNTs [15] SWCNTs (50 nm length)/Au electrode [35] GCE modified with IL/CNTs and gold nanoparticles [11] Aligned CNTs modified with Pt nanoparticles and CdS [13] GCE modified with Si3N4 nanoparticles (this work)
Amperommetry Voltammetry Amperometry Voltammetry Amperometry DPV [c] Voltammetry Amperometry Amperometry Amperometry Amperometry Voltammetry Voltammetry Amperometry Amperometry Voltammetry
0.15–1.2 0.2–1.4 Up to 7.8 Up to 0.7 0.03–5 up to 1.02 0.1 2.1 0.15–10 up to 6 0.5–5.2 up to 20 up to 100 0.002–0.05 0.4–21.2 0.025–8 0.2–2
– – – – 0.024 10 0.05 0.08 0.034 – – – 0.008 0.046 0.0065 0.02
0.3 3.4 0.52 1.018 0.446 13 cm2 3.1 1.92 6.5 cm2 3.96 – – 49 0.045 38.57 cm2 41.41 cm2
3.96 12.6 7.73 – 25.2 4.6 10.1 5 1.3 1.01 9.08 42 2.12 3.8 47.4 –
[a] Glassy carbon electrode; [b] Carbon nanotubes; [c] Differential pulse voltammetry. Electroanalysis 2010, 22, No. 20, 2434 – 2442
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Fig. 4. (A) The first (a) and 100th (b) cyclic voltammograms of the Si3N4/GOx/GC electrode in pH 4 PBS, scan rate 50 mV s1 .(B) Cyclic voltammetric response of the Si3N4/GOx/GC electrode at different pH (from right to left), 2, 4, 6, 8 , 10 and 12 scan rate 200 mV s1. Inset: Variation of standard potentials vs. pH values.
nanoparticles increase the surface area, active point for adsorbing GOx and also make the film more porous for facilitating electron transfer. The working stability of GC/Si3N4/GOx was verified by monitoring the remaining amount of active enzyme after successive sweeps of CVs, 100 cycles with scan rate of 50 mV s1 in potential range 0.0 to 0.45 V (Figure 4A). The peak height and potential of the immobilized enzyme remained nearly unchanged and amount of GOx remain2438
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ing on the electrode surface were almost 95 % of its initial value. In order to study the reproducibility of the biosensor and reliability of fabrication procedure, six GC/Si3N4/ GOx modified electrodes were prepared independently. The relative standard deviation (RSD) value of measured cathodic peak currents was 5 %. Furthermore, the relative standard deviation (RSD) determined by 10 successive assays of a 20 mM glucose sample was 3 %. Good reproducibility and stability of the biosensor can be attributed
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to the high degree of biocompatibility of Si3N4 toward GOx. Cyclic voltammograms of modified electrode were recorded at different buffer solutions (pH 2–12) in the absence of oxygen (Figure 4B). A pair of well defined and stable redox peaks obtained for adsorbed GOx in different pH values. Both reduction and oxidation peak potentials of the FAD/FADH2 redox couple are negatively shifted by increasing the pH values. This response is due to the proton transfer from FAD ( the oxidized quinone form ) to the reduced FADH2 ( the fully reduced hydroquinone form) which is depicted as follow.
þ
ð1Þ
FAD þ 2e þ 2H ! FADH2
The linear regression is E8’ (mV) = 51.52 pH58.2 (R2 = 0.9933), where E8’ is the formal potential [E8’ = (Epa + Epc)/2]. The slope 51.52 is reasonably close to the theoretical value of 58.6 mV pH1 for a reversible two protons and two electron transfer process according to the above reaction. The pH value also affects the redox peak currents of GOx. The peak current is unchanged at pH range 2–7. With a further increase of pH, the peak current decreases gradually, possibly because of the decrease of proton concentration and bioactivity of the immobilized GOx [9].
3.2. Catalytic Activity of GC/Si3N4/GOx Films Figure 5 shows the cyclic voltammetric responses of the GC/Si3N4/GOx biosensor in pH 7 PBS containing 0.2 mM of Fc in the presence different glucose concentration. By introducing glucose to the buffer solution well defined
sigmoidal catalytic waves were observed as a consequence of the GOx catalytic oxidation of glucose. As shown, with increasing glucose concentration anodic responses of the modified electrode increased. The electrocatalytic oxidation of glucose can be described based on the following mechanism [29]. Fc G H Fcþ þ e GOxðFADÞ þ G G
ð2Þ k1
H GOxðFAD GÞ
k1
k
2 GOxðFADH2 Þ þ Gl !
k3 GOxðFADH2 Þ þ 2Fcþ ! GOxðFADÞ þ 2Fc
ð3Þ ð4Þ
Where GOx (FADH2) and GOx (FAD) represent reduced and oxidized forms of glucose oxidase, Fc and Fc + the reduced and oxidized forms of ferrocenemethanol, and G and Gl are b-d-glucose and glucono-d-lactone, respectively. In the absence of mass transport limitations, the plateau current from the ferrocenemethanol-mediated cyclic voltammograms (Ip) is expected to obey the following equation[30]. 1=I p ¼ 1=2FSG ET ð1=k3 ½Fc þ 1=k2 þ 1=kred ½GÞ
ð5Þ
Where F is the Faradays constant, GET the surface concentration of enzyme, S the electrode area, [Fc] the mediator concentration, [G] the glucose concentration in the solution, and kred = k2 k1/(k1+k2). Ip was measured from the background corrected current value by subtracting the glucose free current to glucose contain current at a potential of 0.3 V. Based on the above equation and the known rate constant values, k2 = 700 s1, k3 = 1.2 107 M1 s1, kred = 1.1 104 M1 s1 [31], the surface concen-
Fig. 5. (A) CVs of Si3N4/GOx/electrode in PBS, pH 7 containing 0.2 mM Fc in the absence( a) and presence (b to g) 12, 24, 36, 48 and 60 mM of glucose, scan rate 20 mV s1. Electroanalysis 2010, 22, No. 20, 2434 – 2442
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Fig. 6. CVs of Si3N4/GOx/electrode in PBS, pH 7 (a) deoxygenated, (b) oxygen saturated, (c to p) such as b in the presence different concentration of glucose: 0.2 to 3 mM, scan rate 20 mV s1. Inset: Plot of decreased catalytic peak currents vs. glucose concentration.
tration of GOx deposited at the electrode surface was determined. The coverage of active enzyme GET, was estimated to be 6.3 1013 mol cm2. If we consider that a saturated monolayer of GOx corresponds to a surface concentration of 1.7 1012 mol cm2 [27], the determined GET corresponds to 37 % of a monolayer, which is close to coverage of active GOx immobilized onto silica nanoparticles, 6.0 1013 mol cm2 [32]. The bioactivity of glucose biosensor was evaluated by recording cyclic voltammograms of the GC/Si3N4/GOx electrode in buffer solution saturated with oxygen and containing different concentration of glucose. Figure 6 shows cyclic voltammograms of GC/Si3N4/GOx biosensor in PBS (pH 7) saturated with oxygen in the absence and presence of different concentrations of glucose. Compared with N2-saturated buffer (Figure 6 voltammogram a), a pair of well defined GOx redox peak was also observed , in air saturated buffer in the absence of glucose the cathodic peak current is dramatically increased and anodic peak current is disappeared. Therefore, GC/Si3N4/ GOx modified electrode electrocatalyzed the reduction of oxygen. Upon adding the glucose to the oxygen saturated solution the reduction peak currents decreased (voltammograms c to q in Figure 2B). The GOx catalyzed the oxidation reaction of glucose, which also consumes the dissolved oxygen. The amount of glucose is detected by monitoring the decrease in the reduction peak current of GC/Si3N4/GOx electrode, based on the following equations [33]. GOxðFADÞ þ 2e þ 2Hþ ! GOxðFADH2 Þ in deoxygenated PBS 2440
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ð6Þ
GOxðFADH2 Þ þ O2 ! GOxðFADÞ þ H2 O2 in oxygen saturated PBS
ð7Þ
GOxðFADÞ þ Glucose þ O2 ! GOxðFADH2 Þ þ Glucolactone in oxygen saturated PBS ð8Þ The biosensor exhibited a linear response to glucose in the concentration range 0.2–1.8 mM (n = 9, R2 = 0.9979) with a sensitivity of 41.41 mA mM1 cm2, and detection limit 21 mM. The detection limit, sensitivity and linear range are more better or comparable with other glucose biosensor based on various nanomaterials [4–16] (Table 1). 3.3. Amperometric Detection of Glucose at GC/Si3N4/ GOx Electrode A typical current-time plot of the GC/Si3N4/GOx modified electrode in pH 7 PBS containing 0.2 mM of Fc at a constant potential of 0.3 V during successive additions of glucose (0.6 mM and 25 mM) was recorded (Figure 7). During glucose addition the oxidation current rose steeply and the steady state was achieved within 5 s. The glucose biosensor response vs. glucose concentration is linear over the wide concentration range of 25 mM to 8 mM. The calibration plot over the concentration range of 25–250 mM (10 points) has a slope of 38.57 mA mM1 cm2 (sensitivity), correlation coefficient of 0.9998 and the detection limit of 6.5 mM at signal to noise ratio of 3. These analytical parameters are better
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Fig. 7. Amperometric response of Si3N4/GOx/electrode in PBS, pH 7 (rotation speed 1000 rpm) held at 0.30 V in PBS , pH 7 containing 0.5 mM of ferrocenemethanol, during successive addition of glucose (a) 0.6 mM and (b) 25 mM. Insets: (c and d) Plots of chronoamperometric current vs. glucose concentration. (f) Linear calibration curve for determination of KM. (f) Chronoamperogram of modified electrode for 0.6 mM glucose during a long period time, 1100 s.
than other glucose biosensor have been fabricated based on different nanomaterials [4–16] (Table 1). For glucose concentration higher than 8 mM, a response plateau was observed, showing the characteristics of the Michaelis– Menten kinetic mechanism. The apparent Michaelis– Menten constant (KM), which gives an indication of the enzyme-substrate kinetics, can be obtained from the Lineweaver–Burk equation [34]. 1=I ss ¼ 1=I max þ KM =I max 1=C
ð9Þ
Here, Iss is the steady-state current after the addition of substrate, C is the bulk concentration of substrate and Imax is the maximum current measured under saturated substrate solution. The apparent Michaelis-Menten constant (KM), was calculated to be 7.4 ( 0.2) mM , implying that the GOx/Si3N4 modified glassy carbon electrode exhibits a higher affinity for glucose oxidation. As shown in Figure 7f, the biosensor response remains stable after 20 min continuous operation (unnoticeable change was observed). In addition after storing of the biosensor in 4 8C in dry state the current response, for 1 mM glucose decreased about 5 % of the original value after 10 days. The good stability during measurements and long lifetime of the biosensor may be attributed to the unique nanostructure and the biocompatibility of the novel Si3N4 nanoparticles. Electroanalysis 2010, 22, No. 20, 2434 – 2442
4. Conclusions Silicon nitride nanoparticles is an attractive matrix to immobilize GOx enzyme and exhibits facile , direct electrochemistry of GOx without any electron transfer mediator. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation. The proposed biosensor was used as amperometric glucose biosensor with high sensitivity 38.57 mA mM1 cm2 , low detection limit, 6.5 mM, good stability, short response time (5 s) at wide concentration range; up to 8 mM of glucose. The good stability and high sensitivity of the biosensor indicated that Si3N4, can be used as an attractive material for fabrication of electrochemical biosensors, bioelectronics devices and bioreactors due to direct electrochemistry, high surface area, and unique nanostructure for efficient immobilization of biomolecules.
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