Biosensors and Bioelectronics 71 (2015) 278–285
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A novel ultrasensitive phosphate amperometric nanobiosensor based on the integration of pyruvate oxidase with highly ordered gold nanowires array Edward Ogabiela a, Samuel B. Adeloju a,n, Jiewu Cui b, Yucheng Wu b, Wei Chen c a
NanoScience and Sensor Technology Research Group, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia Laboratory for Functional Nanomaterials and Devices, School of Materials Science and Engineering, Hefei University of Technology, Hefei 230009, Anhui, China c School of Biotechnology & Food Engineering, Hefei University of Technology, Hefei 230009, Anhui, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 11 April 2015 Accepted 11 April 2015 Available online 14 April 2015
A novel phosphate amperometric nanobiosensor, based on an intimate integration of pyruvate oxidase (PyOx) and its cofactors, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), with a highly ordered gold nanowires array (AuNWA) has been developed. The successful integration of PyOx and the co-factors, via crosslinking with bovine serum albumin (BSA) and glutaraldehyde (GLA), onto the AuNWA was confirmed by cyclic voltammetry and amperometry. The resulting nanobiosensor achieved a detection limit of 0.1 mM, a linear concentration range of 12.5–1000 mM, and a sensitivity of 140.3 mA mM 1 cm 2. Notably, the incorporation of the AuNWA reduced the required PyOx concentration by 70–120 fold and the presence of common interferants, such as chloride, sulfate, fluoride, nitrite and nitrate ions did not interfere with phosphate detection. Furthermore, the nanobiosensor demonstrated a very high stability with repeated use over two weeks and was successfully used for the determination of phosphate in water samples with an average recovery of 96.6 74.9%. & 2015 Elsevier B.V. All rights reserved.
Keywords: Phosphate Pyruvate oxidase Gold nanowires array Nanobiosensor Crosslinking Amperometry
1. Introduction The use of nanomaterials, such as metallic nanoparticles, graphene, nanotubes and nanowires has attracted huge interest for fabrication of nanobiosensors for the detection of glucose (Du et al., 2007; Luo et al., 2004), cholesterol (Gopalan et al., 2009), urea (Tiwari et al., 2009), triglycerides (Narang and Pundir, 2011), and pesticides (Simonian et al., 2005). These nanomaterials have unique advantages in the direct wiring of enzymes to electrode surfaces and are capable of retaining their bioactivities due to the high surface areas which readily enable higher enzyme loadings. The presence of these nanomaterials also provides desirable microenvironments, and promotes direct electron transfer between the active sites of the biomolecules and the electrode (Iijima, 1991; Zhu et al., 2012). Although these nanomaterials have been used for fabrication of various nanobiosensors (Baby and Ramaprabhu, 2010; Cui et al., 2014; Rahman et al., 2010; Shao et al., 2010; Zhu et al., 2012), their use for the development of phosphate nanobiosensors has been rather limited. Most of the reported use of nanomaterials for the n
Corresponding author. E-mail address: [email protected] (S.B. Adeloju).
http://dx.doi.org/10.1016/j.bios.2015.04.026 0956-5663/& 2015 Elsevier B.V. All rights reserved.
fabrication of phosphate biosensor has focused to date on the use of nanoparticles (Karthikeyan and Berchmans, 2013; Norouzi et al., 2010). For example, Karthikeyan and Berchmans (2013) immobilized PyOx on a gold substrate functionalized with Cu nanoparticles on a self-assembled monolayer (SAM) of mercapto benzoic acid (MBA). A linear range of 40–350 mM was achieved with a sensitivity of 6 mA mM 1 cm 2 and a detection limit of 0.2 mM. Also, in another study (Norouzi et al., 2010), a glassy carbon electrode modified with PyOx and MWCNTs was used to achieve a detection limit of 0.1 μM and a linear range of 1–100 mM. Nevertheless, it is still possible to achieve substantial improvement in the sensitivity and achievable linear concentration ranges of phosphate nanobiosensors by using nanomaterials that offer larger surface areas, such as nanowires, nanorods and nanowires/ nanotubes array. This view is well supported by the established knowledge that ordered one-dimensional nanomaterials, such as nanowires and nanorods, give better performance than those based on the use of nanoparticles (Wu et al., 2007; Liu et al., 2008; Kong et al., 2009; Zhou et al., 2009; Wang et al., 2009; Yang et al., 2009; Yang et al., 2010). Nanowires (NWs) offer new and unique opportunities for development of novel enzyme-based biosensors because of their ability to act as a “bridge” between the active sites of enzymes and
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the surface of electrodes to promote fast electron transfer (Noor and Krull, 2014; Ariffin et al., 2014; Patolsky et al., 2006). Furthermore, these nanomaterials can also reach the active sites of enzymes. Also, as electrochemical biosensing is surface dependent, the incorporation of NWs enables achievement of larger surface area leading to improvement in performance (Cui et al., 2014; García et al., 2014). For this reason, there has been an increasing interest in using different metallic NWs for biosensors (Bo et al., 2011; García et al., 2014; Hao et al., 2014; Ibupoto et al., 2014; Jamal et al., 2010; Lee et al., 2011; Li et al., 2010; Noor and Krull, 2014; Wang et al., 2010). However, most focus on the use of different NWs for fabrication of biosensors has largely been directed towards glucose biosensors (Cui et al., 2014; Gerola et al., 2014; Wang et al., 2013b, 2013a; Xu et al., 2012; Horng et al., 2009; Jung and Lim, 2013; Rahman et al., 2010). Among the various NWs, AuNWA has many advantages that are beneficial for fabrication of a phosphate nanobiosensor, such as large specific surface area, chemical inertness, biocompatibility, excellent electrical conductivity and good electrochemical activity towards H2O2 (Cui et al., 2014; Guo et al., 2009; Yang et al., 2007). In spite of these potential benefits, the use of AuNWA has never been considered for fabrication of a phosphate nanobiosensors. A key challenge in using AuNWA for fabrication of nanobiosensor is the ability to adequately immobilize the desired enzyme or biomolecule with the nanomaterial. This is critical for achieving ultrasensitive detection of the desired analyte. Although some biosensors have been reported for phosphate detection in water, the approaches used for immobilization of the enzyme may not be suitable for use with AuNWA. Also, there is still a need for further improvement in sensitivity, reliability and linear concentration to permit determination in pristine water samples where phosphate concentrations are very low and still poses a serious challenge for most analytical methods. The achievement of this goal will also enable application to a wider range of samples, including biological and clinical samples. Furthermore, the incorporation of nanomaterials, such as nanowires, can result in substantial reduction of required PyOx concentration and, hence, cost. In the present study, the fabrication of a novel ultrasensitive phosphate amperometric nanobiosensor has been investigated by use of BSA and GLA to achieve an intimate integration of PyOx and its cofactors, TPP and FAD, with a highly ordered AuNWA. Although a mixture of BSA and GLA has been used to immobilize enzymes, it has never been used to immobilize PyOx and co-factors for fabrication of a phosphate nanobiosensor. Detailed investigation of the morphology and features of the AuNWA was therefore necessary and was carried out by field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy and X-ray diffractometry. Furthermore, the performance of the AuNWA–GLA– BSA–FAD–TPP–PyOx nanobiosensor for ultrasensitive amperometric detection of phosphate was investigated by optimizing important parameters, such as GLA, BSA, FAD, TPP, PyOx, PA and buffer concentrations, as well as solution pH. The effect of common interferants such as chloride, fluoride, sulfate, nitrite and nitrate on the nanobiosensor performance was also investigated. Furthermore, the application of the nanobiosensor to the determination of phosphate in pond water was also investigated.
2. Materials and methods 2.1. Chemicals and reagents PyOx (EC. 1.2.3.3; 67 U mg 1) from Aerococcus sp, pyruvic acid, FAD, TPP were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A). Gold chloride trihydrate (HAuCl4 3H2O), Ethylenediaminetetraacetic acid (EDTA), GLA, BSA were obtained from Sinopharm Chemical
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Reagent Co. (Beijing, China). All chemicals were of analytical grade and used as received unless otherwise stated. Milli-Q water (18.2 MOhm cm) was used to prepare all solutions throughout the experiments. Stock solutions of 25% w/v GLA and 20% w/v BSA were prepared and stored in the refrigerator at 4 oC when not in use. All chemicals were of analytical grade, unless otherwise stated. Citrate buffer of 50 mM (pH 7), which contained 1 mM MgCl2 6H2O and 1.5 mM pyruvic acid was used for the amperometric measurements. All solutions were prepared with Milli-Q water. Phosphate stock solution (0.5 M) was stored in the refrigerator and was diluted when necessary to give the required standard concentration. 2.2. Fabrication and characterization of gold nanowires array The AuNWA was grown as described previously (Cui et al. 2014) using a solution which contained 10 g L 1 HAuCl4, 5 g L 1 EDTA, 20 g L 1 K2HPO4 and 160 g L 1 Na2SO3, on the surface of conventional gold electrode directly with the aid of AAO template. Unless otherwise stated, the electrodeposition was carried out with an applied current density of 0.2 mA cm 2. Prior to electrodeposition, a thin gold film was sputtered onto one side of the AAO template by vacuum sputter coater to act as working electrode. After electrodeposition, AAO template was etched away by utilizing 1 M NaOH to expose gold nanowire arrays, and then the arrays were rinsed thoroughly with Milli-Q water and ethanol to remove the residual NaOH. All electrochemical depositions were carried out on a galvanostat–potentiostat with a three electrode system at room temperature. Morphology and microstructure of AuNWAs were characterized by field emission scanning electron microscopy (FESEM, Hitachi S 4800). The fabricated AuNWA was fixed onto platinum disk electrode with the aid of a conductive carbon tape. 2.3. Water sample The AuNWA–GLA–BSA–FAD–TPP–PyOx biosensor was used to determine phosphate concentration in pond water samples collected from a pond in Hefei University of Technology, Hefei (Anhui, China).
3. Results and discussion 3.1. Synthesis, morphology and characterization of AuNWA The AuNWA used in this study was synthesized with the aid of an AAO template which was fabricated by using a two-step anodization process with an applied potential of 45 V for 4 h. The pore diameter and depth of the template is dependent on the anodization voltage and anodization time, respectively. An extended anodization time is useful for achieving adequate pore depth and uniformity of the AAO membrane. Fig. 1A shows the SEM image for the fabricated AAO templates with pores in a parallel arrangement. An AAO template with a diameter of 80 nm was used for the fabrication of the AuNWA. Fig. 1B shows the top view of the synthesized AuNWA with a length of 450 nm standing straight after dissolving the aluminum oxide membrane for 10 min in 1 M sodium hydroxide. The insert (1) in Fig. 1B shows the side view of the AuNWA standing erect. This diameter and the length of the nanowires are ideal for achieving high enzyme and co-factor loadings for phosphate detection. The EDX spectroscopy profile of the AuNWA, shown in Fig. 1C, indicates that the nanowires comprised mainly of gold (100%). This is further corroborated by the XRD profile for the AuNWA in Fig. 1D which clearly revealed diffraction peaks of Au (111) and Au (200) corresponding with 2θ ¼38.32o and 44.41o, respectively. The confirmation of the presence of gold in the NWA ensures that all the benefits usually
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Fig.1. Characterization of AuNWA by FESEM, energy dispersive X-ray spectroscopy and X-ray diffractometry. (A) Top-view of AAO template by FESEM, (B) Highly ordered gold nanowire arrays by FESEM, Insert (1) of AuNWA standing erect with height of 450 nm, (C) Energy dispersive X-ray spectroscopy of the AuNWA, (D) X-ray-diffraction profile of the AuNWA.
associated with AuNWA, such as large specific surface area, chemical inertness, biocompatibility, excellent electrical conductivity and good electrochemical activity towards H2O2 (Cui et al., 2014; Guo et al., 2009; Yang et al., 2007), will be readily realised for optimum fabrication of the phosphate nanobiosensor. 3.2. Electrochemical characterization and amperometric detection of phosphate Fig. 2A illustrates the substantial improvement in the cyclic voltammetric behavior observed for the Fe(CN)64 /3 redox system with the use of AuNWA compared to the use of a conventional gold disk electrode. The current magnitudes obtained for the reduction and oxidation peaks clearly demonstrate that the electroactive surface area of the AuNWA is at least 6 times larger than that of the gold disk electrode. Evidently, the use of AuNWA gave an increased surface area which enabled a much improved
electron transfer. These observations indicate that the use of the AuNWA for fabrication of a phosphate nanobiosensor should enable a substantial improvement in analytical performances. However, the realization of such an outcome will depend on the effective optimization of several factors associated with the integration of PyOx and co-factors with the AuNWA. The influence of scan rates on the cathodic and anodic peaks obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx electrode is shown in Fig. 2B. Both the anodic and cathodic peak currents increased linearly with increasing scan rate from 20 to 120 mV/s, indicating that the electrode reactions involved surface confined process (Rahman et al., 2006). Also, the peak separation of the redox couple increased with increasing scan rates, suggesting that the electrode process was quasi-reversible (Cui et al., 2014). Evidently, the reversibility of the electrode process was affected by the incorporation of BSA, GLA, PyOx and other components with the AuNWA. Nevertheless, Fig. 2C shows that the cathodic and
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Fig. 2. Electrochemical characterization of AuNWA and phosphate detection with the nanobiosensor. (A) Cyclic voltammograms obtained with (a) conventional gold electrode, and (b) AuNWA in 0.1 M KCl which contained 10 mM K3Fe(CN)6. Scan rate: 100 mV/s. (B) Cyclic voltammograms of AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor in 0.05 M citrate buffer solution containing 0.1 M KCl and 10 mM K3Fe(CN)6 at different scan rates (a) 20 mV/s, (b) 40 mV/s, (c) 80 mV/s, (d) 80 mV/s, ( e ) 100 mV/s and (f) 120 mV/s. (C) Relationship between peak current density and square root of the scan rate from Fig. 2B.
anodic current densities are directly proportional to the square root of the scan rate and, hence, indicate that a diffusion-controlled process was involved at the AuNWA–GLA–BSA–FAD–TPP– PyOx electrode surface. The operational principle for the detection of phosphate with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor is illustrated in Scheme 1. The corresponding equations are given below:
Pyruvate + phosphate + 02 PyOx
→ acetylphosphate + CO2 + H2 O2
(1)
H2O2 - 2H þ þO2 þ 2e
(2)
where PyOx, in the presence of cofactors, such as TPP and FAD, catalyses the oxidative decarboxylation of pyruvate to produce acetylphosphate, CO2 and H2O2. The resulting H2O2 reaches the AuNWA surface, where it is oxidized to give amperometric response which is directly proportional to the phosphate concentration. The high electrochemical activity of AuNWA towards H2O2 will ensure optimum amperometric response for the detection of phosphate (Cui et al., 2014). Also, the large active surface area provided by AuNWA will enable an increased PyOx loading
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Pyruvic acid + phosphate + O2 Acetylphosphate + H2O2 +CO2
H2O2 H2O + O2 PyOx FAD TPP Scheme 1. Schematic illustration of AuNWA–GLA–BSA–FAD–TPP–PyOx phosphate nanobiosensor.
and, hence, reduce the H2O2 path between PyOx active sites and the AuNWA surface. Consequently, an enhancement in the electron transfer will be realised. However, to ensure that the integration of PyOx with AuNWA is effective and reliable for ultrasensitive amperometric detection of phosphate, it was necessary to further characterize the composite nanobiosensor and investigate the influence of key parameters on its analytical performances. Fig. 3a shows the cyclic voltammograms obtained in 0.1 M citrate buffer with the nanobiosensors after integration of AuNWA with PyOx and one or more cofactors. Evidently, the shape and the current magnitude of the voltammograms changed with the inclusion of TPP and FAD. The progressive change with the inclusion of one and both co-factors serves to confirm their inclusion and retention within the BSA–GLA layer. Also, the increasing current magnitude indicates that the AuNWA–GLA–BSA–FAD–TPP–PyOx nanocomposite becomes more conductive with the inclusion of the co-factors. The cofactors FAD and TPP are essential for the catalytic activity of PyOx and, hence, their presence is necessary for an effective performance of the phosphate nanobiosensor. The electrochemical behavior of two phosphate biosensors constructed with a conventional Au disk electrode and the AuNWA was compared in citrate buffer to ascertain the improvement gained by the inclusion of the nanomaterial. Fig. 3b shows that, compared to the Au–BSA–GLA–FAD–TPP–PyOx biosensor, the current magnitude obtained with the AuNWA–GLA–BSA–FAD– TPP–PyOx nanobiosensor was substantially larger due to the large surface area provided by AuNWA and the increased PyOx loading. Furthermore, Fig. 3c shows that both the anodic and cathodic currents obtained with AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor increased with increasing phosphate concentration. These results indicate that the amperometric detection of phosphate with the nanobiosensor can be achieved by application of a potential within 0 and 300 mV or within þ200 and þ600 mV. Similar results were reported by Rahman et al. (2006) for the covalent immobilization of PyOx onto a conducting polymer layer which composed of nanoparticles on a glassy carbon electrode used for amperometric detection of phosphate ion and also by Karthikeyan and Berchmans (2013) who immobilized PyOx on a gold substrate functionalized with Cu nanoparticles which was stabilized on a self-assembled monolayer (SAM) of mercapto benzoic acid for amperometric detection of phosphate. Fig. 3d shows the phosphate amperometric responses obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor compared to those obtained with a conventional Au–GLA–BSA–FAD– TPP–PyOx biosensor. Evidently, the phosphate responses obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor were
at least three times more sensitive than those obtained with the conventional electrode. Furthermore, much more resolved phosphate amperometric responses were obtained with the nanobiosensor. 3.3. Optimization of the phosphate nanobiosensor The nanobiosensor was further optimized for amperometric detection of phosphate by investigating the influence of applied potential, solution pH, buffer, PyOx and pyruvic acid concentrations. The results are presented in Fig. S1 and discussed in details in the supplementary information. In summary, the optimum conditions were: applied potential 125 mV, pH 7, 50 mM citrate buffer, 1.5 mM pyruvic acid and 10 U/mL PyOx. FAD and TPP are also required for the catalytic activity of PyOx, otherwise the phosphate response is severely diminished in their absence. As a homo-tetrameric enzyme, each PyOx subunit (62 kDa) binds to FAD noncovalently and also binds TPP loosely, while Mg2 þ is the metal cation for the reaction which is catalysed by TPP (Ogabiela and Adeloju, 2014). PyOx in the presence of TPP and FAD catalyse the decaboxylation of pyruvate to yield acetylephosphate, CO2 and H2O2 as shown in Eq. (1). Consequently, the phosphate response obtained with the nanobiosensor in this study increased with increasing FAD concentration and reached an optimum with the addition of 5 mM FAD. Beyond this concentration, the phosphate response decreased due to the presence of excessive FAD. The optimum FAD concentration in this study is lower than that reported in our previous study (Ogabiela and Adeloju, 2014) and lower than 10 mM used by Kwan et al. (2005b) to fabricate potentiometric and amperometric phosphate biosensors, respectively. Gilbert et al. (2010, 2011) used 20 mM FAD in their studies, which was four times higher than used in our present study. The phosphate response obtained with the nanobiosensor also increased with increasing TPP concentration and gave optimum response with the addition of 200 mM TPP. This concentration is close to that used in another study (Gilbert et al., 2011), but it is higher than the 70 mM TPP used in our previous study (Ogabiela and Adeloju, 2014) and in another study (Kwan et al., 2005b). The higher TPP required was due to the increased surface area and increased PyOx loading on the AuNWA. Further increase in TPP concentration resulted in a decrease in the phosphate response due to the presence of excessive TPP concentration (Arai et al. ,1999; Kubo et al., 1991; Ogabiela and Adeloju, 2014). A FAD concentration of 5 mM and TPP concentration of 200 mM were therefore used for subsequent investigations. The integration of PyOx with the AuNWA was also optimized by investigating the influence of BSA concentration, GLA concentration
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Fig. 3. Cyclic voltammograms obtained (a) with different AuNWA electrodes: (i) AuNWA–GLA–BSA–PyOx, (ii) AuNWA–GLA–BSA–TPP–PyOx, and (iii) AuNWA–GLA–BSA– FAD–TPP–PyOx nanobiosensor in 0.05 M citrate buffer; (b) Au–GLA–BSA–FAD–TPP–PyOx and AuNWA–GLA–BSA–FAD–TPP–PyOx; and (c) AuNWA–GLA–BSA–FAD–TPP–PyOx biosensor in the presence of increasing phosphate concentration. Scan rate: 100 mV/s and (d) comparison of phosphate amperometric responses obtained with conventional gold electrode and AuNWA–GLA–BSA–FAD–TPP–PyOx biosensors.
and the drying time. The results of the optimization are presented in Fig. S2 and discussed in details in the supplementary information. The optimum conditions achieved for these three parameters are 2.4% w/v BSA, 4.5% v/v GLA and a drying time of 30 min. 3.4. Analytical performance, interference study and stability The bioactivity of the PyOx and co-factors integrated with the AuNWA via crosslinking BSA and GLA was investigated by calculating the apparent Michaelis–Menten constant Kapp m based on the use of the Lineweaver–Burk equation: 1/iss ¼(Kapp m /imax) (1/C)þ1/imax where iss is the steady state current after addition of phosphate, imax is the maximum current, and C is phosphate concentration in the measurement solution. From the Lineweaver–Burk plot, the apparent Michaelis–Menten constant was calculated to be 9.0 mM,
which was much lower than 14.6 mM reported recently (Sitte, 2013), but was higher than 2 mM reported by Gilbert et al. (2010). The low Kapp m achieved in this study is indicative of the strong affinity between the phosphate in solution and the immobilized PyOx in the nanobiosensor. The selectivity of the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor was evaluated with successive addition of 500 mM phosphate, 500 mM Cl , 500 mM SO42 , 500 mM F , 500 mM NO3 , and 500 mM NO2 , followed by another 500 mM phosphate. All interferants had no effect on the phosphate amperometric response. The usual concentrations of these anions in river, lakes and pond water are lower and, therefore, not likely to interfere with phosphate determination in water samples with the nanobiosensor. Fig. 4 shows that the phosphate amperometric responses obtained with the AuWNA–GLA–BSA–FAD–TPP–PyOx nanobiosensor increased with increasing addition of phosphate. Evidently, a wide
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Fig. 4. Typical amperometric responses and linear concentration range obtained with AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor.
Table 1 Phosphate recovery in pond water sample with the nanobiosensor. Concentration added (mM)
Concentration found (mM)
Recovery (%)
0 50 250 500 750
8.0 7 1.2 56.17 4.2 249.3 7 6.1 490.47 11.6 736.2 7 26.0
– 96.2 78.2 96.5 72.7 96.5 72.3 97.0 76.2
phosphate concentrations, as indicated in Table 1. Evidently excellent recoveries of the phosphate spikes in the water samples were achieved with the nanobiosensor, ranging from 96.2% to 97.0% with a RSD of 2.3% to 8.2% (n ¼3). The average recovery for all spiked phosphate concentrations was 96.6 74.9%. The phosphate concentration obtained with the nanobiosensor in the pond water sample was 8.0 71.2 mM (n¼ 3).
n¼ 3.
4. Conclusions
linear concentration range was obtained with the nanobiosensor between 12.5 mM and 1000 mM (R2 ¼0.992) phosphate. This linear range is significantly wider than 40–350 mM achieved with CuNPs (Karthikeyan and Berchmans (2013)) and 1–100 mM achieved with MWCNTs (Norouzi et al., 2010). Furthermore, a sensitivity of 140.3 mA mM 1 cm 2 achieved with the nanobiosensor is also significantly better than 6 mA mM 1 cm 2 achieved with CuNPs (Karthikeyan and Berchmans, 2013). Both of these observations clearly reflect the superior performance of the AuNWA integrated with PyOX and co-factors via crosslinking with BSA and GLA for phosphate amperometric detection. The achieved detection limit (3s) of 0.1 mM was calculated as dl ¼3s/m, where s was the standard deviation of background current, m was the sensitivity of the biosensors. This detection limit is better than 0.2 mM achieved with CuNPs (Karthikeyan and Berchmans, 2013) and comparable with 0.1 mM achieved with MWCNTs (Norouzi et al., 2010). It is also worth noting that 100% of the original phosphate amperometric response was maintained over two weeks of repeated use with in-between storage in the refrigerator in 50 mM citrate buffer solution. The excellent retention of the original phosphate amperometric response over this period clearly reflects the effectiveness of the integrated PyOx and co-factors with the AuNWA. It also demonstrates the excellent retention of PyOx and co-factors within the BSA–GLA gel despite repeated use and storage in the buffer. The nanobiosensor was also successfully employed for reliable determination of phosphate in water samples. The reliability of the nanobiosensor was verified for phosphate determination by a recovery study in pond water samples spiked with different
The successful fabrication and utilization of a novel amperometric AuWNA–GLA–BSA–FAD–TPP–PyOx nanobiosensor has been demonstrated for an ultrasensitive detection of phosphate. The resulting nanobiosensor gave the best linear concentration range, reported to date, of 12.5–1000 mM for phosphate, as well as the best sensitivity of 140.3 mA mM 1 cm 2, and one of the best detection limit of 0.1 mM for phosphate detection with any nanobiosensor. Furthermore, the incorporation of AuNWA in the nanobiosensor enabled a 70–120-fold reduction in the required PyOx concentration compared to those used in earlier studies (Ikebukuro, et al. 1996; Kwan et al., 2005a; Kwan et al., 2005b; Mak et al., 2003) and, consequently, enabled more economical use of PyOx for fabrication of the phosphate nanobiosensor. The biosensor also demonstrated an excellent stability, maintaining its original phosphate amperometric response for two weeks with repeated use. In addition, it demonstrated a very high selectivity against major interferants such as chloride, sulfate, fluoride, nitrate and nitrite ions and was successfully used for phosphate determination in pond water samples with an excellent average recovery of 96.67 4.9%.
Acknowledgment One of the authors (Edward Ogabiela) acknowledges the scholarships and travel grant provided by Monash Institute of Graduate Research, and the support provided for his research visit by the School of Material Science and Engineering, Hefei University of Technology, Hefei, China.
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.026.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel ultrasensitive phosphate amperometric nanobiosensor based on the integration of pyruvate oxidase with highly ordered gold nanowires array Edward Ogabiela a, Samuel B. Adeloju a,n, Jiewu Cui b, Yucheng Wu b, Wei Chen c a
NanoScience and Sensor Technology Research Group, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia Laboratory for Functional Nanomaterials and Devices, School of Materials Science and Engineering, Hefei University of Technology, Hefei 230009, Anhui, China c School of Biotechnology & Food Engineering, Hefei University of Technology, Hefei 230009, Anhui, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 11 April 2015 Accepted 11 April 2015 Available online 14 April 2015
A novel phosphate amperometric nanobiosensor, based on an intimate integration of pyruvate oxidase (PyOx) and its cofactors, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), with a highly ordered gold nanowires array (AuNWA) has been developed. The successful integration of PyOx and the co-factors, via crosslinking with bovine serum albumin (BSA) and glutaraldehyde (GLA), onto the AuNWA was confirmed by cyclic voltammetry and amperometry. The resulting nanobiosensor achieved a detection limit of 0.1 mM, a linear concentration range of 12.5–1000 mM, and a sensitivity of 140.3 mA mM 1 cm 2. Notably, the incorporation of the AuNWA reduced the required PyOx concentration by 70–120 fold and the presence of common interferants, such as chloride, sulfate, fluoride, nitrite and nitrate ions did not interfere with phosphate detection. Furthermore, the nanobiosensor demonstrated a very high stability with repeated use over two weeks and was successfully used for the determination of phosphate in water samples with an average recovery of 96.6 74.9%. & 2015 Elsevier B.V. All rights reserved.
Keywords: Phosphate Pyruvate oxidase Gold nanowires array Nanobiosensor Crosslinking Amperometry
1. Introduction The use of nanomaterials, such as metallic nanoparticles, graphene, nanotubes and nanowires has attracted huge interest for fabrication of nanobiosensors for the detection of glucose (Du et al., 2007; Luo et al., 2004), cholesterol (Gopalan et al., 2009), urea (Tiwari et al., 2009), triglycerides (Narang and Pundir, 2011), and pesticides (Simonian et al., 2005). These nanomaterials have unique advantages in the direct wiring of enzymes to electrode surfaces and are capable of retaining their bioactivities due to the high surface areas which readily enable higher enzyme loadings. The presence of these nanomaterials also provides desirable microenvironments, and promotes direct electron transfer between the active sites of the biomolecules and the electrode (Iijima, 1991; Zhu et al., 2012). Although these nanomaterials have been used for fabrication of various nanobiosensors (Baby and Ramaprabhu, 2010; Cui et al., 2014; Rahman et al., 2010; Shao et al., 2010; Zhu et al., 2012), their use for the development of phosphate nanobiosensors has been rather limited. Most of the reported use of nanomaterials for the n
Corresponding author. E-mail address: [email protected] (S.B. Adeloju).
http://dx.doi.org/10.1016/j.bios.2015.04.026 0956-5663/& 2015 Elsevier B.V. All rights reserved.
fabrication of phosphate biosensor has focused to date on the use of nanoparticles (Karthikeyan and Berchmans, 2013; Norouzi et al., 2010). For example, Karthikeyan and Berchmans (2013) immobilized PyOx on a gold substrate functionalized with Cu nanoparticles on a self-assembled monolayer (SAM) of mercapto benzoic acid (MBA). A linear range of 40–350 mM was achieved with a sensitivity of 6 mA mM 1 cm 2 and a detection limit of 0.2 mM. Also, in another study (Norouzi et al., 2010), a glassy carbon electrode modified with PyOx and MWCNTs was used to achieve a detection limit of 0.1 μM and a linear range of 1–100 mM. Nevertheless, it is still possible to achieve substantial improvement in the sensitivity and achievable linear concentration ranges of phosphate nanobiosensors by using nanomaterials that offer larger surface areas, such as nanowires, nanorods and nanowires/ nanotubes array. This view is well supported by the established knowledge that ordered one-dimensional nanomaterials, such as nanowires and nanorods, give better performance than those based on the use of nanoparticles (Wu et al., 2007; Liu et al., 2008; Kong et al., 2009; Zhou et al., 2009; Wang et al., 2009; Yang et al., 2009; Yang et al., 2010). Nanowires (NWs) offer new and unique opportunities for development of novel enzyme-based biosensors because of their ability to act as a “bridge” between the active sites of enzymes and
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the surface of electrodes to promote fast electron transfer (Noor and Krull, 2014; Ariffin et al., 2014; Patolsky et al., 2006). Furthermore, these nanomaterials can also reach the active sites of enzymes. Also, as electrochemical biosensing is surface dependent, the incorporation of NWs enables achievement of larger surface area leading to improvement in performance (Cui et al., 2014; García et al., 2014). For this reason, there has been an increasing interest in using different metallic NWs for biosensors (Bo et al., 2011; García et al., 2014; Hao et al., 2014; Ibupoto et al., 2014; Jamal et al., 2010; Lee et al., 2011; Li et al., 2010; Noor and Krull, 2014; Wang et al., 2010). However, most focus on the use of different NWs for fabrication of biosensors has largely been directed towards glucose biosensors (Cui et al., 2014; Gerola et al., 2014; Wang et al., 2013b, 2013a; Xu et al., 2012; Horng et al., 2009; Jung and Lim, 2013; Rahman et al., 2010). Among the various NWs, AuNWA has many advantages that are beneficial for fabrication of a phosphate nanobiosensor, such as large specific surface area, chemical inertness, biocompatibility, excellent electrical conductivity and good electrochemical activity towards H2O2 (Cui et al., 2014; Guo et al., 2009; Yang et al., 2007). In spite of these potential benefits, the use of AuNWA has never been considered for fabrication of a phosphate nanobiosensors. A key challenge in using AuNWA for fabrication of nanobiosensor is the ability to adequately immobilize the desired enzyme or biomolecule with the nanomaterial. This is critical for achieving ultrasensitive detection of the desired analyte. Although some biosensors have been reported for phosphate detection in water, the approaches used for immobilization of the enzyme may not be suitable for use with AuNWA. Also, there is still a need for further improvement in sensitivity, reliability and linear concentration to permit determination in pristine water samples where phosphate concentrations are very low and still poses a serious challenge for most analytical methods. The achievement of this goal will also enable application to a wider range of samples, including biological and clinical samples. Furthermore, the incorporation of nanomaterials, such as nanowires, can result in substantial reduction of required PyOx concentration and, hence, cost. In the present study, the fabrication of a novel ultrasensitive phosphate amperometric nanobiosensor has been investigated by use of BSA and GLA to achieve an intimate integration of PyOx and its cofactors, TPP and FAD, with a highly ordered AuNWA. Although a mixture of BSA and GLA has been used to immobilize enzymes, it has never been used to immobilize PyOx and co-factors for fabrication of a phosphate nanobiosensor. Detailed investigation of the morphology and features of the AuNWA was therefore necessary and was carried out by field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy and X-ray diffractometry. Furthermore, the performance of the AuNWA–GLA– BSA–FAD–TPP–PyOx nanobiosensor for ultrasensitive amperometric detection of phosphate was investigated by optimizing important parameters, such as GLA, BSA, FAD, TPP, PyOx, PA and buffer concentrations, as well as solution pH. The effect of common interferants such as chloride, fluoride, sulfate, nitrite and nitrate on the nanobiosensor performance was also investigated. Furthermore, the application of the nanobiosensor to the determination of phosphate in pond water was also investigated.
2. Materials and methods 2.1. Chemicals and reagents PyOx (EC. 1.2.3.3; 67 U mg 1) from Aerococcus sp, pyruvic acid, FAD, TPP were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A). Gold chloride trihydrate (HAuCl4 3H2O), Ethylenediaminetetraacetic acid (EDTA), GLA, BSA were obtained from Sinopharm Chemical
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Reagent Co. (Beijing, China). All chemicals were of analytical grade and used as received unless otherwise stated. Milli-Q water (18.2 MOhm cm) was used to prepare all solutions throughout the experiments. Stock solutions of 25% w/v GLA and 20% w/v BSA were prepared and stored in the refrigerator at 4 oC when not in use. All chemicals were of analytical grade, unless otherwise stated. Citrate buffer of 50 mM (pH 7), which contained 1 mM MgCl2 6H2O and 1.5 mM pyruvic acid was used for the amperometric measurements. All solutions were prepared with Milli-Q water. Phosphate stock solution (0.5 M) was stored in the refrigerator and was diluted when necessary to give the required standard concentration. 2.2. Fabrication and characterization of gold nanowires array The AuNWA was grown as described previously (Cui et al. 2014) using a solution which contained 10 g L 1 HAuCl4, 5 g L 1 EDTA, 20 g L 1 K2HPO4 and 160 g L 1 Na2SO3, on the surface of conventional gold electrode directly with the aid of AAO template. Unless otherwise stated, the electrodeposition was carried out with an applied current density of 0.2 mA cm 2. Prior to electrodeposition, a thin gold film was sputtered onto one side of the AAO template by vacuum sputter coater to act as working electrode. After electrodeposition, AAO template was etched away by utilizing 1 M NaOH to expose gold nanowire arrays, and then the arrays were rinsed thoroughly with Milli-Q water and ethanol to remove the residual NaOH. All electrochemical depositions were carried out on a galvanostat–potentiostat with a three electrode system at room temperature. Morphology and microstructure of AuNWAs were characterized by field emission scanning electron microscopy (FESEM, Hitachi S 4800). The fabricated AuNWA was fixed onto platinum disk electrode with the aid of a conductive carbon tape. 2.3. Water sample The AuNWA–GLA–BSA–FAD–TPP–PyOx biosensor was used to determine phosphate concentration in pond water samples collected from a pond in Hefei University of Technology, Hefei (Anhui, China).
3. Results and discussion 3.1. Synthesis, morphology and characterization of AuNWA The AuNWA used in this study was synthesized with the aid of an AAO template which was fabricated by using a two-step anodization process with an applied potential of 45 V for 4 h. The pore diameter and depth of the template is dependent on the anodization voltage and anodization time, respectively. An extended anodization time is useful for achieving adequate pore depth and uniformity of the AAO membrane. Fig. 1A shows the SEM image for the fabricated AAO templates with pores in a parallel arrangement. An AAO template with a diameter of 80 nm was used for the fabrication of the AuNWA. Fig. 1B shows the top view of the synthesized AuNWA with a length of 450 nm standing straight after dissolving the aluminum oxide membrane for 10 min in 1 M sodium hydroxide. The insert (1) in Fig. 1B shows the side view of the AuNWA standing erect. This diameter and the length of the nanowires are ideal for achieving high enzyme and co-factor loadings for phosphate detection. The EDX spectroscopy profile of the AuNWA, shown in Fig. 1C, indicates that the nanowires comprised mainly of gold (100%). This is further corroborated by the XRD profile for the AuNWA in Fig. 1D which clearly revealed diffraction peaks of Au (111) and Au (200) corresponding with 2θ ¼38.32o and 44.41o, respectively. The confirmation of the presence of gold in the NWA ensures that all the benefits usually
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Fig.1. Characterization of AuNWA by FESEM, energy dispersive X-ray spectroscopy and X-ray diffractometry. (A) Top-view of AAO template by FESEM, (B) Highly ordered gold nanowire arrays by FESEM, Insert (1) of AuNWA standing erect with height of 450 nm, (C) Energy dispersive X-ray spectroscopy of the AuNWA, (D) X-ray-diffraction profile of the AuNWA.
associated with AuNWA, such as large specific surface area, chemical inertness, biocompatibility, excellent electrical conductivity and good electrochemical activity towards H2O2 (Cui et al., 2014; Guo et al., 2009; Yang et al., 2007), will be readily realised for optimum fabrication of the phosphate nanobiosensor. 3.2. Electrochemical characterization and amperometric detection of phosphate Fig. 2A illustrates the substantial improvement in the cyclic voltammetric behavior observed for the Fe(CN)64 /3 redox system with the use of AuNWA compared to the use of a conventional gold disk electrode. The current magnitudes obtained for the reduction and oxidation peaks clearly demonstrate that the electroactive surface area of the AuNWA is at least 6 times larger than that of the gold disk electrode. Evidently, the use of AuNWA gave an increased surface area which enabled a much improved
electron transfer. These observations indicate that the use of the AuNWA for fabrication of a phosphate nanobiosensor should enable a substantial improvement in analytical performances. However, the realization of such an outcome will depend on the effective optimization of several factors associated with the integration of PyOx and co-factors with the AuNWA. The influence of scan rates on the cathodic and anodic peaks obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx electrode is shown in Fig. 2B. Both the anodic and cathodic peak currents increased linearly with increasing scan rate from 20 to 120 mV/s, indicating that the electrode reactions involved surface confined process (Rahman et al., 2006). Also, the peak separation of the redox couple increased with increasing scan rates, suggesting that the electrode process was quasi-reversible (Cui et al., 2014). Evidently, the reversibility of the electrode process was affected by the incorporation of BSA, GLA, PyOx and other components with the AuNWA. Nevertheless, Fig. 2C shows that the cathodic and
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Fig. 2. Electrochemical characterization of AuNWA and phosphate detection with the nanobiosensor. (A) Cyclic voltammograms obtained with (a) conventional gold electrode, and (b) AuNWA in 0.1 M KCl which contained 10 mM K3Fe(CN)6. Scan rate: 100 mV/s. (B) Cyclic voltammograms of AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor in 0.05 M citrate buffer solution containing 0.1 M KCl and 10 mM K3Fe(CN)6 at different scan rates (a) 20 mV/s, (b) 40 mV/s, (c) 80 mV/s, (d) 80 mV/s, ( e ) 100 mV/s and (f) 120 mV/s. (C) Relationship between peak current density and square root of the scan rate from Fig. 2B.
anodic current densities are directly proportional to the square root of the scan rate and, hence, indicate that a diffusion-controlled process was involved at the AuNWA–GLA–BSA–FAD–TPP– PyOx electrode surface. The operational principle for the detection of phosphate with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor is illustrated in Scheme 1. The corresponding equations are given below:
Pyruvate + phosphate + 02 PyOx
→ acetylphosphate + CO2 + H2 O2
(1)
H2O2 - 2H þ þO2 þ 2e
(2)
where PyOx, in the presence of cofactors, such as TPP and FAD, catalyses the oxidative decarboxylation of pyruvate to produce acetylphosphate, CO2 and H2O2. The resulting H2O2 reaches the AuNWA surface, where it is oxidized to give amperometric response which is directly proportional to the phosphate concentration. The high electrochemical activity of AuNWA towards H2O2 will ensure optimum amperometric response for the detection of phosphate (Cui et al., 2014). Also, the large active surface area provided by AuNWA will enable an increased PyOx loading
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Pyruvic acid + phosphate + O2 Acetylphosphate + H2O2 +CO2
H2O2 H2O + O2 PyOx FAD TPP Scheme 1. Schematic illustration of AuNWA–GLA–BSA–FAD–TPP–PyOx phosphate nanobiosensor.
and, hence, reduce the H2O2 path between PyOx active sites and the AuNWA surface. Consequently, an enhancement in the electron transfer will be realised. However, to ensure that the integration of PyOx with AuNWA is effective and reliable for ultrasensitive amperometric detection of phosphate, it was necessary to further characterize the composite nanobiosensor and investigate the influence of key parameters on its analytical performances. Fig. 3a shows the cyclic voltammograms obtained in 0.1 M citrate buffer with the nanobiosensors after integration of AuNWA with PyOx and one or more cofactors. Evidently, the shape and the current magnitude of the voltammograms changed with the inclusion of TPP and FAD. The progressive change with the inclusion of one and both co-factors serves to confirm their inclusion and retention within the BSA–GLA layer. Also, the increasing current magnitude indicates that the AuNWA–GLA–BSA–FAD–TPP–PyOx nanocomposite becomes more conductive with the inclusion of the co-factors. The cofactors FAD and TPP are essential for the catalytic activity of PyOx and, hence, their presence is necessary for an effective performance of the phosphate nanobiosensor. The electrochemical behavior of two phosphate biosensors constructed with a conventional Au disk electrode and the AuNWA was compared in citrate buffer to ascertain the improvement gained by the inclusion of the nanomaterial. Fig. 3b shows that, compared to the Au–BSA–GLA–FAD–TPP–PyOx biosensor, the current magnitude obtained with the AuNWA–GLA–BSA–FAD– TPP–PyOx nanobiosensor was substantially larger due to the large surface area provided by AuNWA and the increased PyOx loading. Furthermore, Fig. 3c shows that both the anodic and cathodic currents obtained with AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor increased with increasing phosphate concentration. These results indicate that the amperometric detection of phosphate with the nanobiosensor can be achieved by application of a potential within 0 and 300 mV or within þ200 and þ600 mV. Similar results were reported by Rahman et al. (2006) for the covalent immobilization of PyOx onto a conducting polymer layer which composed of nanoparticles on a glassy carbon electrode used for amperometric detection of phosphate ion and also by Karthikeyan and Berchmans (2013) who immobilized PyOx on a gold substrate functionalized with Cu nanoparticles which was stabilized on a self-assembled monolayer (SAM) of mercapto benzoic acid for amperometric detection of phosphate. Fig. 3d shows the phosphate amperometric responses obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor compared to those obtained with a conventional Au–GLA–BSA–FAD– TPP–PyOx biosensor. Evidently, the phosphate responses obtained with the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor were
at least three times more sensitive than those obtained with the conventional electrode. Furthermore, much more resolved phosphate amperometric responses were obtained with the nanobiosensor. 3.3. Optimization of the phosphate nanobiosensor The nanobiosensor was further optimized for amperometric detection of phosphate by investigating the influence of applied potential, solution pH, buffer, PyOx and pyruvic acid concentrations. The results are presented in Fig. S1 and discussed in details in the supplementary information. In summary, the optimum conditions were: applied potential 125 mV, pH 7, 50 mM citrate buffer, 1.5 mM pyruvic acid and 10 U/mL PyOx. FAD and TPP are also required for the catalytic activity of PyOx, otherwise the phosphate response is severely diminished in their absence. As a homo-tetrameric enzyme, each PyOx subunit (62 kDa) binds to FAD noncovalently and also binds TPP loosely, while Mg2 þ is the metal cation for the reaction which is catalysed by TPP (Ogabiela and Adeloju, 2014). PyOx in the presence of TPP and FAD catalyse the decaboxylation of pyruvate to yield acetylephosphate, CO2 and H2O2 as shown in Eq. (1). Consequently, the phosphate response obtained with the nanobiosensor in this study increased with increasing FAD concentration and reached an optimum with the addition of 5 mM FAD. Beyond this concentration, the phosphate response decreased due to the presence of excessive FAD. The optimum FAD concentration in this study is lower than that reported in our previous study (Ogabiela and Adeloju, 2014) and lower than 10 mM used by Kwan et al. (2005b) to fabricate potentiometric and amperometric phosphate biosensors, respectively. Gilbert et al. (2010, 2011) used 20 mM FAD in their studies, which was four times higher than used in our present study. The phosphate response obtained with the nanobiosensor also increased with increasing TPP concentration and gave optimum response with the addition of 200 mM TPP. This concentration is close to that used in another study (Gilbert et al., 2011), but it is higher than the 70 mM TPP used in our previous study (Ogabiela and Adeloju, 2014) and in another study (Kwan et al., 2005b). The higher TPP required was due to the increased surface area and increased PyOx loading on the AuNWA. Further increase in TPP concentration resulted in a decrease in the phosphate response due to the presence of excessive TPP concentration (Arai et al. ,1999; Kubo et al., 1991; Ogabiela and Adeloju, 2014). A FAD concentration of 5 mM and TPP concentration of 200 mM were therefore used for subsequent investigations. The integration of PyOx with the AuNWA was also optimized by investigating the influence of BSA concentration, GLA concentration
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Fig. 3. Cyclic voltammograms obtained (a) with different AuNWA electrodes: (i) AuNWA–GLA–BSA–PyOx, (ii) AuNWA–GLA–BSA–TPP–PyOx, and (iii) AuNWA–GLA–BSA– FAD–TPP–PyOx nanobiosensor in 0.05 M citrate buffer; (b) Au–GLA–BSA–FAD–TPP–PyOx and AuNWA–GLA–BSA–FAD–TPP–PyOx; and (c) AuNWA–GLA–BSA–FAD–TPP–PyOx biosensor in the presence of increasing phosphate concentration. Scan rate: 100 mV/s and (d) comparison of phosphate amperometric responses obtained with conventional gold electrode and AuNWA–GLA–BSA–FAD–TPP–PyOx biosensors.
and the drying time. The results of the optimization are presented in Fig. S2 and discussed in details in the supplementary information. The optimum conditions achieved for these three parameters are 2.4% w/v BSA, 4.5% v/v GLA and a drying time of 30 min. 3.4. Analytical performance, interference study and stability The bioactivity of the PyOx and co-factors integrated with the AuNWA via crosslinking BSA and GLA was investigated by calculating the apparent Michaelis–Menten constant Kapp m based on the use of the Lineweaver–Burk equation: 1/iss ¼(Kapp m /imax) (1/C)þ1/imax where iss is the steady state current after addition of phosphate, imax is the maximum current, and C is phosphate concentration in the measurement solution. From the Lineweaver–Burk plot, the apparent Michaelis–Menten constant was calculated to be 9.0 mM,
which was much lower than 14.6 mM reported recently (Sitte, 2013), but was higher than 2 mM reported by Gilbert et al. (2010). The low Kapp m achieved in this study is indicative of the strong affinity between the phosphate in solution and the immobilized PyOx in the nanobiosensor. The selectivity of the AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor was evaluated with successive addition of 500 mM phosphate, 500 mM Cl , 500 mM SO42 , 500 mM F , 500 mM NO3 , and 500 mM NO2 , followed by another 500 mM phosphate. All interferants had no effect on the phosphate amperometric response. The usual concentrations of these anions in river, lakes and pond water are lower and, therefore, not likely to interfere with phosphate determination in water samples with the nanobiosensor. Fig. 4 shows that the phosphate amperometric responses obtained with the AuWNA–GLA–BSA–FAD–TPP–PyOx nanobiosensor increased with increasing addition of phosphate. Evidently, a wide
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Fig. 4. Typical amperometric responses and linear concentration range obtained with AuNWA–GLA–BSA–FAD–TPP–PyOx nanobiosensor.
Table 1 Phosphate recovery in pond water sample with the nanobiosensor. Concentration added (mM)
Concentration found (mM)
Recovery (%)
0 50 250 500 750
8.0 7 1.2 56.17 4.2 249.3 7 6.1 490.47 11.6 736.2 7 26.0
– 96.2 78.2 96.5 72.7 96.5 72.3 97.0 76.2
phosphate concentrations, as indicated in Table 1. Evidently excellent recoveries of the phosphate spikes in the water samples were achieved with the nanobiosensor, ranging from 96.2% to 97.0% with a RSD of 2.3% to 8.2% (n ¼3). The average recovery for all spiked phosphate concentrations was 96.6 74.9%. The phosphate concentration obtained with the nanobiosensor in the pond water sample was 8.0 71.2 mM (n¼ 3).
n¼ 3.
4. Conclusions
linear concentration range was obtained with the nanobiosensor between 12.5 mM and 1000 mM (R2 ¼0.992) phosphate. This linear range is significantly wider than 40–350 mM achieved with CuNPs (Karthikeyan and Berchmans (2013)) and 1–100 mM achieved with MWCNTs (Norouzi et al., 2010). Furthermore, a sensitivity of 140.3 mA mM 1 cm 2 achieved with the nanobiosensor is also significantly better than 6 mA mM 1 cm 2 achieved with CuNPs (Karthikeyan and Berchmans, 2013). Both of these observations clearly reflect the superior performance of the AuNWA integrated with PyOX and co-factors via crosslinking with BSA and GLA for phosphate amperometric detection. The achieved detection limit (3s) of 0.1 mM was calculated as dl ¼3s/m, where s was the standard deviation of background current, m was the sensitivity of the biosensors. This detection limit is better than 0.2 mM achieved with CuNPs (Karthikeyan and Berchmans, 2013) and comparable with 0.1 mM achieved with MWCNTs (Norouzi et al., 2010). It is also worth noting that 100% of the original phosphate amperometric response was maintained over two weeks of repeated use with in-between storage in the refrigerator in 50 mM citrate buffer solution. The excellent retention of the original phosphate amperometric response over this period clearly reflects the effectiveness of the integrated PyOx and co-factors with the AuNWA. It also demonstrates the excellent retention of PyOx and co-factors within the BSA–GLA gel despite repeated use and storage in the buffer. The nanobiosensor was also successfully employed for reliable determination of phosphate in water samples. The reliability of the nanobiosensor was verified for phosphate determination by a recovery study in pond water samples spiked with different
The successful fabrication and utilization of a novel amperometric AuWNA–GLA–BSA–FAD–TPP–PyOx nanobiosensor has been demonstrated for an ultrasensitive detection of phosphate. The resulting nanobiosensor gave the best linear concentration range, reported to date, of 12.5–1000 mM for phosphate, as well as the best sensitivity of 140.3 mA mM 1 cm 2, and one of the best detection limit of 0.1 mM for phosphate detection with any nanobiosensor. Furthermore, the incorporation of AuNWA in the nanobiosensor enabled a 70–120-fold reduction in the required PyOx concentration compared to those used in earlier studies (Ikebukuro, et al. 1996; Kwan et al., 2005a; Kwan et al., 2005b; Mak et al., 2003) and, consequently, enabled more economical use of PyOx for fabrication of the phosphate nanobiosensor. The biosensor also demonstrated an excellent stability, maintaining its original phosphate amperometric response for two weeks with repeated use. In addition, it demonstrated a very high selectivity against major interferants such as chloride, sulfate, fluoride, nitrate and nitrite ions and was successfully used for phosphate determination in pond water samples with an excellent average recovery of 96.67 4.9%.
Acknowledgment One of the authors (Edward Ogabiela) acknowledges the scholarships and travel grant provided by Monash Institute of Graduate Research, and the support provided for his research visit by the School of Material Science and Engineering, Hefei University of Technology, Hefei, China.
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.04.026.
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