Modified Tin Oxide Based Bioelectrode for Reagentless Detection of Uric Acid

A reagentless uric acid biosensor has been realized using Copper implanted tin oxide thin film (Cu:SnO2) based matrix. The biosensing characteristics of implanted matrix have been studied using the electrochemical impedance spectroscopy and cyclic voltammetry. The prepared matrix (Cu:SnO2), because of the presence of Cu, possess redox properties so that the electron transfer from enzyme to the electrode could be accomplished without using any external mediator in the electrolyte. The developed uric acid biosensor exhibits a high sensitivity of about 0.93 mA/ mM and a linear variation in current response over a concentration range from 0.05 to 1.0 mM of uric acid besides high shelf life (~20 weeks). The Michaelis-Menten kinetic parameter (Km) is found to be relatively very low (0.12 mM), which indicates the high affinity of the fabricated bioelectrode towards the uric acid (analyte). The results highlight the importance of implanted Cu:SnO2 thin film as an attractive matrix for the realization of reagentless biosensors towards uric acid. Citation: Arora K, Tomar M, Gupta V (2017) Modified Tin Oxide Based Bioelectrode for Reagentless Detection of Uric Acid. J Biosens Bioelectron 8: 246. doi: 10.4172/2155-6210.1000246


Introduction
Uric acid is present in number of biological fluids including urine and blood serum and is associated with the presence of diseases such as gout, hyperuricemia, cholesterol, arthritis, renal, neurological, kidney diseases etc. Thus, efficient detection of uric acid in human blood is of great interest to the researchers. Many reports are available on the detection of uric acid [1,2], but the presence of external mediator in the electrolyte hinders its usage for implantable biosensor. Limited work has been carried out on the development of reagentless uric acid biosensors, where no external mediating element is needed. Table 1 summarizes the work carried out on uric acid biosensors by various workers using different matrices. Various matrices have been utilized for the detection of uric acid such as APTES Bis sulfosuccinimide/ITO [3], gold electrode/polystyrene [4], carbon felt based H 2 O 2 [5], poly N-isopropyl acrylamide MWCNT-Ch/poly(amidoamine)/DNA/gold electrode [6], Polyaniline [7], screen printed electrode [8] and so on. However, the low sensitivity and poor shelf life of the biosensors are major concern. Metal oxide thin film-based bioelectrodes are shown to be advantageous. Tin oxide (SnO 2 ), a wide band gap semiconductor (E g =3.87 eV), has multifunctional properties for broad range of applications including transparent electronics, gas sensors, acoustic devices, UV photo detectors, etc.
Recently SnO 2 is being explored for biosensing applications owing to its excellent charge transfer capability, good biocompatibility, low cost, abundance in nature etc. [9]. However, the non-redox property of SnO 2 demands an external mediator in the electrolyte, which hinders its application for realizing an integrated biosensor. Few attempts have been made towards tailoring the properties of metal oxides by introducing suitable metal dopants [10]. The metal ions having multiple valence states such as Fe, Cu, Mn, V etc. can be incorporated to induce redox properties in the host metal oxide matrix [11]. However, most of the reports on doped metal oxide matrix are using chemical route which lead to the preparation of bioelectrodes with poor stability [12]. The rapid decay in the response of these bioelectrodes with time may be due to poor incorporation of metal dopants at the lattice sites of host oxide matrix and poor quality of deposited films. Implantation is an effective method to incorporate the foreign elements in the metal oxides and to tailor its properties. Few reports are available on incorporating redox species into the ZnO matrix using ion implantation of Fe for reagentless detection of glucose [13]. Surprisingly, no efforts are being made to modify the electronic properties of SnO 2 by implanting it with suitable metals that have multiple oxidation states especially for detection of uric acid. Amongst different metals, copper (Cu) has multi-electron oxidation states and is attractive due to natural abundance, low cost, non-toxic and good electrochemical activity [6,14]. The impantation of Cu in SnO 2 may induce the redox property besides enhancing the electron transfer property for possible biosensing application.
In this paper, the SnO 2 thin films were prepared by RF sputtering technique and Cu implanted SnO 2 matrix has been utilized for development of reagentless uric acid biosensor. The biosensor is found to be exhibiting excellent response characteristics important for use in implantable biosensor chip.
MΩ cm was used for the preparation of aqueous solutions. Different concentrations of uric acid solution (0.05-1.0 mM) and solution of o-dianisidine (1%) dye were freshly prepared in de-ionized water.

Cu implanted SnO 2 thin film based electrode
A 100 nm thin Pt layer was deposited on the surface of corning glass slide over an area of 2 cm × 1 cm by RF sputtering technique using a 3 inch diameter metal Pt target (99.99% pure) under 100% Ar gas ambient and a power of 100 W. A buffer layer (~ 10 nm) of titanium (Ti) was sputter coated prior to Pt deposition under the same growth condition onto the glass slide using a Ti target (99.99% pure) to improve the adhesion of Pt on the glass slide. SnO 2 thin film was deposited on to Pt/Ti coated Corning glass slide using RF magnetron sputtering technique on an area of 1cm×1cm while the remaining 1 cm × 1 cm area was protected using shadow mask for electrical connections. A 2 inch diameter metallic tin target (99.999% pure) was sputtered in a reactive gas mixture (70% Ar and 30% O 2 ) at an rf power of 30 W and sputtering pressure of 20 mTorr. The thickness of SnO 2 thin film was maintained at 180 nm. Subsequently, Copper (Cu) was implanted in as-grown SnO 2 thin film at room temperature at low energy (15 keV) to a fluence of 10 15 and 10 16 cm -2 . The ion implantation was carried out on low energy ion implanter -150 kV accelerator at the Australian National University, Australia. The biosensor electrode for detection of uric acid has been prepared on Cu implanted SnO 2 matrix and the fabrication steps are shown schematically in Figure 1.

Characterization techniques
The structural and optical properties of the developed Cu implanted SnO 2 matrix were studied using an X-ray diffractometer (Bruker D8 Discover) and a double beam Uv-visible spectrophotometer (Perkin Elmer, Lambda 25-35-45) respectively. Implantation of Cu into SnO 2 matrix is confirmed by energy-dispersive X-ray spectroscopy (EDX) analysis. Modification of surface morphology before and after immobilization of the uricase were investigated using an atomic force microscope (Veeco DICP2) in non-contact mode, a scanning electron microscope and a fourier transform infrared spectrophotometer (Perkin Elmer (Model Spectrum BX). Micro images obtained from AFM were analyzed by SPM Lab Analysis software. Electrochemical measurements were carried out on a potentiostat/galvanostat/ZRA (Gamry reference using a conventional three-electrode electrochemical cell with Ag/AgCl as reference electrode, platinum (Pt) foil as counter electrode and uricase/Cu:SnO 2 /Pt/Ti/glass as working electrode. The enzyme activity of bio-electrode was studied using UV-visible spectrophotometer (Perkin Elmer, Lambda 35). The thickness of deposited thin film was determined using surface profiler (Dektak 150A). PBS solution (10 ml, 50 mM, 0.9% NaCl) without any mediating species was used as electrolyte.

X-Ray Diffraction (XRD) studies
The XRD studies of as-deposited SnO 2 films shows the growth of amorphous film, which became polycrystalline after a post deposition   annealing treatment at 300°C in air for 2 h. The XRD spectrum for the annealed SnO 2 film deposited on glass slide is shown in Supplementary  Figure 1a. Well defined reflection peaks corresponding to (110), (101), (200) and (211) planes of rutile SnO 2 were observed in the XRD pattern [15], indicating that the annealed SnO 2 thin film is polycrystalline. The average crystallite size of the SnO 2 film was calculated from the dominant (101) XRD peak using the Debye Scherrer's formula and is found to be about 9 nm.  Figure 1). The crystallite size of implanted samples is found to be almost same (10 nm) as that of pure SnO 2 film.

Energy-dispersive X-ray spectroscopy (EDX) analysis of Cu implanted SnO 2 matrix
EDX spectra of bare SnO 2 and Cu implanted (10 16 cm -2 fluence) SnO 2 films deposited on Pt coated Si wafer are shown in Figure 2. The presence of both Sn and O can be easily seen in the EDX spectra along with the weak peaks corresponding to Pt and Si, which are due to the underneath Pt electrode and Si wafer, respectively. The additional peaks observed corresponding to the Cu in the EDX spectra of Cu implanted SnO 2 film (Cu:SnO 2 ), clearly confirm the presence of Cu in the implanted SnO 2 matrix. electrolyte without any redox speciclearly seen that no redox peak is observed in the CV spectra for SnO 2 /Pt/Ti/glass electrode in PBS solution in the entire measured range of voltage (-0.5 V-0.3 V) because SnO 2 does not have any redox couple of its own. Hence, in the absence of any mediator in the electrolyte solution SnO 2 does not show any redox peak. On the other hand, the CV spectrum of Cu:SnO 2 /Pt/glass electrode, clearly shows well defined redox peaks in a mediator-free PBS buffer which is attributed to valence transformation of copper from Cu 1+ to Cu 2+ and vice-versa, thus resulting in accelerated electron transfer. These results indicate that Cu is responsible for the reversible redox property in the electrode and serves as the electron transfer channel. The value of peak oxidation current obtained for Cu implanted SnO 2 /Pt/Ti/glass matrix was found to be much higher in comparison to that reported for other ion implanted metal oxide matrices for biosensing such as Fe:ZnO [16]. It may be clearly seen from inset (a) of Figure 3 that the electrode implanted at a fluence of 10 16 cm -2 gives higher peak oxidation current (0.72 mA) than at lower fluence and hence chosen for further studies.  film was rough (average roughness ~10 nm) with fine microstructure having uniformly distributed small size grains, and is beneficial for the efficient loading of enzyme on its surface. Upon immobilization of uricase on the surface of implanted matrix, clusters of uricase biomolecules forming a globular structure of much bigger size (~ 2 µm) are seen Figure 4b. This confirms the successful immobilization of enzyme (uricase) onto the surface of Cu:SnO 2 /Pt/Ti/glass electrode.

FTIR studies
The prepared Cu implanted SnO 2 films were characterized by FTIR spectroscopy before and after immobilization of enzyme and the corresponding spectra are shown in Supplementary Figures 2a and 2b, respectively. The sharp and well defined absorption peak observed at around 606 cm −1 in Supplementary Figure 2a

Electrochemical impedance spectroscopy (EIS)
Electrochemical impedance spectroscopy (EIS) analysis has been carried out using a three-electrode cell with the SnO 2 /Pt/Ti/glass and Cu:SnO 2 /Pt/T/glass electrodes as working electrode, platinum (Pt) foil as counter electrode and Ag/AgCl as reference electrode in PBS solution containing [K 3 Fe(CN) 6 ] 3-/4as the mediator in the frequency range 100 kHz to 10 mHz. Supplementary Figure 3

Sensing response characteristics
It is known that the pH of the electrolyte solution changes the activity of the biomolecules to a great extent. CV response of the prepared uricase/Cu:SnO 2 /Pt/Ti/glass bio-electrode was investigated at various pH levels (6.0 to 8.0) of the PBS buffer and the variation of the peak oxidation current (I pa ) with pH is shown in the inset of Figure 3. The peak oxidation current (I pa ) was found to increase with an increase in pH from 6.0 to 7.0 having a maximum value of 750 µA and decreases thereafter with further increase in pH. Therefore, the pH of the PBS solution was kept at 7.0 for amperrometric studies. The CV spectra were recorded for uricase/Cu:SnO 2 /Pt/Ti/glass bioelectrode at varying scan rates from 100 to 600 mV/s as shown in Supplementary Figure 4. Both the oxidation (I pa ) and reduction (I pc ) peak currents were found to increase linearly with the increase in scan rate from 100 to 600 mV/s (inset of Supplementary Figure 4). The linear relationship between the peak currents (I pa or I pc ) and scan rate (ν) demonstrates that the electrochemical reaction of immobilized uricase on Cu implanted SnO 2 electrode is a surface-controlled process. Furthermore, the redox potentials corresponding to the anodic and cathodic peak currents shift respectively towards the higher potential and lower potential with  Figure 4). The observed variation in redox potential confirms that the reaction is quasi-reversible.
The surface concentration of ionic species per unit area for uricase/ Cu implanted SnO 2 /Pt/Ti/glass bio-electrode is estimated using Brown-Anson model. The estimated value of surface concentration of electroactive uricase on the surface of the prepared bioelectrode is found to be about 5.42 × 10 -5 mole/cm 2 which is greater in comparison to the corresponding values reported by various workers for uric acid biosensors using other metal oxide matrices [5,6]. The high surface concentration obtained in the present work confirms that the prepared bioelectrode offers high enzyme loading. This is attributed to the preparation of implanted matrix with desired rough surface morphology. The high enzyme loading is expected to provide the enhanced sensitivity of the developed biosensor. The results indicate that Cu implantation is effective not only in enhancing the electron communication property of SnO 2 matrix, but also in increasing the electroactive surface area of the matrix.
The sensing response of the bioelectrode (uricase/Cu:SnO 2 /Pt/ Ti/glass) has been obtained using the CV spectra recorded with increasing concentration of uric acid in the PBS electrolyte ( Figure 5). The physiological range of uric acid in human serum is between 0.13 to 0.46 mM. Thus, the concentration of uric acid (analyte) was varied from 0.05 to 1.0 mM in the electrolyte solution. It may be observed from Supplementary Figure 5 that the oxidation peak current increases linearly from 0.17 mA to 1.09 mA with an increase in uric acid concentration from 0.05 to 1.0 mM. The CV responses were used to draw a calibration curve for the uricase/Cu:SnO 2 /Pt/Ti/glass electrode on interaction with the varying concentration of uric acid as shown in the inset (a) of Figure 5. It may be clearly seen from the calibration curve that the uricase/Cu:SnO 2 /Pt/glass electrode exhibits good linearity for the sensing of uric acid in a wide range from 0.05 mM to 1.00 mM with a correlation coefficient of 0.99. Sensitivity of the uricase/Cu:SnO 2 / Pt/glass bioelectrode is calculated from the slope of the calibration curve and is found to be about 0.93 mA/mM with a low limit of detection (LOD) of 0.06 mM. Sensitivity of the bioelectrode towards uric acid is found to be higher compared to the corresponding values reported by various workers for other metal oxide matrices (Table 1). Furthermore, the bioelectrode is found to exhibit fast response time of about 4 seconds due to the fast electron transfer between the active sites of enzyme and electrode. The affinity of uricase immobilized on the surface of the Cu:SnO 2 film towards uric acid is determined from the value of Michaelis-Menten kinetic parameter (K m ) estimated from Hanes plot as shown in the inset (b) of Figure 5. The estimated value of K m is found to be about 0.12 mM which is lower than values reported by other workers for uric acid biosensors ( Table 1). The low value of K m indicates that the immobilized uricase in the present work has high affinity towards uric acid.
To carry out the photometric enzyme assay, the bioelectrode was Where A is the difference in absorbance before and after incubation, V is the total volume (=3.17 cm 3 ), ε is the millimolar extinction coefficient for o-dianisidine (7.5 for o-dianisidine at 500 nm), t is the reaction time (2 mins.), and s is the surface area of the electrode (1.0 × 1.0 cm 2 ). The value of apparent enzyme activity of the immobilized uricase on the Cu:SnO 2 /Pt/Ti/glass electrode is about 5.01 × 10 -2 unit/ cm 2 . This value is much higher than that obtained by other workers indicating that our bioelectrode provides a better platform and hence more units of uricase are actively working per unit electrode surface area [6,19,7].
The selectivity of our bioelectrode is studied by recording the value of peak oxidation current in CV response on addition of  Figure 5). It may be inferred that the bioelectrode is highly selective towards the uric acid and hence the sensing response is highly reliable. The shelf life studies of the bioelectrode shows that it retains more than 90% of activity even after 20 weeks as shown in Supplementary Figure 6. The enhanced sensing response characteristics are attributed to the preparation of a highly stable and biocompatible Cu implanted SnO 2 thin film matrix in the present study [20][21][22][23][24][25][26][27][28].

Real samples analysis
The prepared uricase/Cu:SnO 2 /Pt/glass biosensor has been exploited for the analysis of uric acid in the real biological fluid, 10 human sera samples were analyzed and the % recovery and precision were calculated for spiked samples. After the addition of the sera samples in the electrolyte solution, the peak oxidation current values have been noted from the CV measurements. Uric acid concentration in serum was then extrapolated from the standard calibration curve between uric acid concentration (mM) and current (inset of Figure 5). Electrochemical sensing technique in the present work has been used to corroborate results obtained by commercial spectrophotometric method in the clinical laboratory. To validate Accuracy of the prepared electrode has been validated by determining the content of uric acid in serum samples and has been compared with that measured by commercial spectrophotometric method in the clinical laboratory. Table 2 summarizes the results obtained during the present investigation. The results indicate that the uric acid analysis in sera with the developed biosensor agrees well with the spectrophotometric data. Recovery tests were also performed in order to demonstrate the accuracy and precision of the prepared biosensor by adding a known concentration of uric acid (0.50 mM) in the already tested serum samples and then estimating the concentration of added uric acid ( Table 2). The performed recovery tests confirm that the fabricated biosensor is accurate and precise. The performance of the prepared biosensor system is comparable with the commercially accepted methods. For in-vivo application of the biosensor in clinical practice, the requirements of biocompatibility, linearity, sensitivity, specificity, accuracy and long-term stability were very well fulfilled by uricase/ Cu:SnO 2 /Pt/Ti/glass based biosensor.

Conclusion
A novel reagentless uric acid biosensor based on efficient transfer of electron from the immobilized enzyme onto the electrode via implanted Cu in SnO 2 matrix is developed. The uricase/Cu:SnO 2 /Pt/ glass bioelectrode exhibits enhanced sensitivity (0.93 mA/mM) with good linearity over a wide detection range (0.05 to1.0 mM) in the absence of any external mediator in the PBS solution. The low K m value (0.12 mM) obtained for the bioelectrode indicates high affinity of uricase immobilized on the surface of Cu:SnO 2 matrix towards uric acid. The apparent enzyme activity is about 5.01 × 10 -2 unit/cm 2 . The biosensor exhibits very high stability with a shelf-life of about 20 weeks. The observed results confirmed that Cu implanted SnO 2 matrix is attractive for realization of efficient uric acid biosensor with enhanced sensitivity, good selectivity and high stability.