Influences of Mg Doping on the Electrochemical Performance of TiO<sub>2</sub> Nanodots Based Biosensor Electrodes

Al-Furjan, M. S. H.; Cheng, Kui; Weng, Wenjian

doi:https://doi.org/10.1155/2014/965821

Advances in Materials Science and Engineering

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Research Article | Open Access

Volume 2014 | Article ID 965821 | https://doi.org/10.1155/2014/965821

Influences of Mg Doping on the Electrochemical Performance of TiO₂ Nanodots Based Biosensor Electrodes

M. S. H. Al-Furjan,¹Kui Cheng,¹and Wenjian Weng¹

Academic Editor: Hossein Moayedi

Received19 Nov 2014

Accepted15 Dec 2014

Published29 Dec 2014

Abstract

Electrochemical biosensors are essential for health monitors to help in diagnosis and detection of diseases. Enzyme adsorptions on biosensor electrodes and direct electron transfer between them have been recognized as key factors to affect biosensor performance. TiO₂ has a good protein adsorption ability and facilitates having more enzyme adsorption and better electron transfer. In this work, Mg ions are introduced into TiO₂ nanodots in order to further improve electrode performance because Mg ions are considered to have good affinity with proteins or enzymes. Mg doped TiO₂ nanodots on Ti substrates were prepared by spin-coating and calcining. The effects of Mg doping on the nanodots morphology and performance of the electrodes were investigated. The density and size of TiO₂ nanodots were obviously changed with Mg doping. The sensitivity of 2% Mg doped TiO₂ nanodots based biosensor electrode increased to 1377.64 from 897.8 µA mM⁻¹ cm⁻² and its decreases to 0.83 from 1.27 mM, implying that the enzyme achieves higher catalytic efficiency due to better affinity of the enzyme with the Mg doped TiO₂. The present work could provide an alternative to improve biosensor performances.

1. Introduction

Electrochemical biosensors are widely investigated and applied in areas related to health monitors, environmental areas, and pharmaceutical and industrial fields [1–5]. Among them, hydrogen peroxide (H₂O₂) biosensors attract more and more attention because H₂O₂ is involved in most of biological events and intercellular pathways and is the by-product of oxidases such as cholesterol oxidase, lactate oxidase, and glutamate oxidase. Among various techniques, the electrochemical approach shows many advantages such as high sensitivity, low cost, rapid response, and simplicity [6].

In electrochemical sensors, direct electron transfer between the enzymes and electrodes has been identified as one of the key factors to determine their performance [7]. Since the direct electron transfer between the enzyme and the bare electrode is very difficult to take place [8], a mediator is usually required to establish a bridge between enzyme and electrode; thus, proper design of electrodes is an effective way to control the direct electron transfer [9]. TiO₂ is a wide band semiconductor with good chemical stability and biocompatibility, and it is reported that the modified electrodes with nanostructured mediator could accelerate the direct electron transfer rate [10, 11]. Hence, nanostructured TiO₂ with high surface area and high surface activity should be a good mediator with a strong protein adsorption ability that favors the electron transfer between enzymes and electrode [12]. There are many studies for doped TiO₂ with several approaches in order to modify and enhance the efficiency of the TiO₂ in various applications [13–17]. For biosensor applications, the doped TiO₂ provides more effective interaction area and more feasible electron transfer interface to support amperometric response of the electrode [18]. Mg ion has been doped in TiO₂ and it showed that the ion could improve the efficiency of TiO₂ in the medical applications [19] due to good affinity of Mg ions with proteins.

Based on our previous work on TiO₂ nanodots based biosensor electrode [20], in this work, we adopted Mg doping and attempted to improve performances of TiO₂ nanodots based H₂O₂ biosensor electrodes through strengthening direct electron transfer with the aid of good affinity of Mg doped TiO₂ nanodots with the enzyme. The influences of Mg doping on TiO₂ nanodots and performance of the resulting biosensors were characterized and discussed.

2. Experimental Design

2.1. Preparation and Characterization of Mg Doped TiO₂ Nanodots Films on Ti Substrate

Titanium (Ti) foils with purity of 99.99% and thickness of 0.1 mm were used. The foils were cut into small substrates with dimensions of (2 × 1) cm and then ultrasonically cleaned in ethanol, deionized water, and acetone (1 : 1 : 1). After rinsing with deionized water and ethanol, the substrates were dried under room temperature. A phase-separation-induced self-assembly approach was used to prepare TiO₂ nanodots on Ti substrate [21]. Briefly, the precursor sol was prepared with ethanol solution of magnesium chloride hexahydrate (MgCl₂·6H₂O, Hushi Chemical Reagent, AR), acetylacetone (AcAc, Lingfeng Chemical Reagent, AR, >99%), polyvinyl pyrrolidone (PVP, K30, Sinopharm Chemical Reagent, AR, >99%), deionized water, and titanium tetrabutoxide (TBOT, Sinopharm Chemical Reagent, CP, >98%). The molar ratio of H₂O : TBOT : AcAc was 1 : 1 : 0.3. Table 1 shows the concentrations of Mg, TBOT, and PVP in the precursor sols for different samples. The homogeneous precursor sol was spin-coated with 30 L onto the Ti substrates at 7500 rpm for 50 s. Then the samples were calcinated in the air at 500°C for 90 min. A field-emission scanning electron microscope (FESEM) (Hitachi, S-4800) was used to observe the morphology of TiO₂ nanodots film.

2.2. Preparation of H₂O₂ Biosensors

Horseradish peroxidase enzyme (HRP) was selected because HRP is available with high sensitivity and purity, and it was used in the fabrication of biosensors for accurate and reliable determination of H₂O₂ [20, 22–25]. HRP was purchased from Aladdin Reagent (250 U mg⁻¹) and stored at 4°C. Nafion (5 wt%) was bought from Sigma-Aldrich, and 0.5 wt% Nafion solution was prepared to be used for immobilizing HRP enzyme and was stored in dark place at room temperature. 0.1 M PBS was prepared and it consisted of NaH₂PO₄, Na₂HPO₄, NaCl, and H₂O. The HCl and NaOH were used to adjust the pH of the PBS. The PBS solution was deoxygenated by bubbling pure N₂ gas for 30 min prior to use. The solution of H₂O₂ (hydrogen peroxide 30%, AR) was freshly prepared. The other chemicals employed in our experiments were of analytical grade and were used as supplied. The distilled deionized water was used for solutions.

Mg doped into TiO₂ nanodots film was sealed with epoxy except 2.5 × 2.5 mm²; it was left as measuring area. The physical adsorption method was used to immobilize the HRP enzyme on the electrode surface, where 6 L HRP solution was dropped on the electrode surface and left to dry at room temperature. The HRP solution was prepared by dissolving HRP in 0.01 M PBS (phosphate buffer solution with pH = 7.4 [26, 27]) in order to obtain 0.01 g mL⁻¹ solution. Finally, 4 L of 0.5 wt% Nafion solution was dropped onto the biosensor surface to protect the enzyme and make the biosensor be biocompatible, because Nafion has been used as an immobilization matrix for the enzyme and to keep the stability of the biosensor for long term [28], and then left it to dry at room temperature (Figure 1). The Nafion/HRP/Mg-TND/Ti electrodes were washed after storing at 4°C for 1 day [26, 27, 29]. The modified Nafion/HRP/Mg-TND/Ti electrodes were stored when not being in use at 4°C.

2.3. H₂O₂ Biosensors Characterization

The modified electrodes were tested by using CHI 660D electrochemical workstation. 0.1 M phosphate buffer solutions were prepared with various pH (5.0, 6.0, 6.5, 7.0, 7.4, and 8.0) in order to select the optimum pH value of the PBS that can show high performance for the biosensor. The performance of the present biosensor electrodes with pH response was shown to be highest at pH 7.0; therefore, pH value of BPS was set at 7.0 for measuring the electrode performances. The cyclic voltammetry techniques were used to test the modified electrodes in PBS 20 mL (PBS, 0.1 M at 25°C and purged with pure nitrogen for 30 min in order to remove the oxygen) and cycled by applying a voltage in the range between −0.2 V and −0.8 V. The amperometric technique was carried out on the biosensors, whereas various applied potential values were applied from −0.4 V to −0.9 V in order to find the optimum applied voltage for the biosensor that give high current response and therefore −0.8 V was selected as optimum applied potential value.

3. Results and Discussion

3.1. Microstructures of Mg Doped TiO₂ Nanodots Based Electrodes

After Mg was doped, the resulting films had similar nanodot morphology; it indicates that the addition of Mg ions in the 0~6 mol% range could not have influence on Marangoni effect induced phase separation during spin-coating, which induces nanodot morphology [21]. However, Mg doping was shown to have changes in density and size of TiO₂ nanodots on Ti (Figure 2). Compared with undoped TiO₂ nanodots, the density of TiO₂ nanodots was inversely proportional to Mg concentration, and the size of TiO₂ nanodots was proportional to the Mg concentration (Table 2). This change could be attributed to the fact that the spherical phase separation in spun thin liquid layer is influenced by chloride anions from Mg precursor. For observation of an individual TiO₂ nanodot under TEM, it was found that the distribution of Mg element in TiO₂ nanodots was homogenous (Figure 3), which favors adsorbing proteins or enzymes.

(a) Mg-TND-2

(b) Mg-TND-4

(c) Mg-TND-6

(a)

(b)

(c)

3.2. Electrochemical Behaviors of Mg Doped TiO₂ Nanodots Based Biosensor Electrodes

Figure 4 shows the cyclic voltammetric curve of Mg doped TiO₂ nanodots based biosensor electrode, indicating that a typical reaction mechanism of hydrogen peroxide biosensor occurred as follows [30, 31]: For the biosensor electrodes with different Mg doping amount, all had a small pair of redox peaks, and Nafion/HRP/Mg-TND-4/Ti and Nafion/HRP/TND-6/Ti biosensor electrodes (curves (a) and (b)) had smaller cathodic peak potential than Nafion/HRP/Mg-TND-2/Ti biosensor electrode (curve (c)), implying that Nafion/HRP/Mg-TND-2/Ti improved well the electrochemical response of HRP enzyme [32]. The better voltammetric response could result from better establishment in electron transfer between the electrode surface and HRP in the Nafion/HRP/Mg-TND-2/Ti electrode.

The anodic peak potential and the cathodic peak potential of Nafion/HRP/Mg-TND-2/Ti were found at −0.335 V and −0.473 V, respectively (curve (c)). The peak to peak separation was found to be 0.138 V. When 100 M H₂O₂ into 0.1 M PBS was added, the Nafion/HRP/TND-3/Ti biosensor electrode was shown to have a good response (curve (d)), indicating that a strong electrocatalytic activity appears in the reduction reaction of H₂O₂. Also the direct electron transfer rate in Nafion/HRP/Mg-TND-2/Ti is shown to be faster than that of the undoped biosensor electrode. This could be attributed to good function of the immobilized HRP on Mg doped TiO₂ nanodots.

Moreover, the CV curves of Nafion/HRP/Mg-TND-2/Ti biosensor electrode were obtained with various scan rates. It was found that the reduction peak current increased linearly with increasing scan rate (from 0.1 to 0.5 V·s⁻¹) in 0.1 M PBS with pH = 7.0 (Figure 5(b)). The increase of cathodic peaks is ascribed to occurrence of an enhanced electrochemical reaction of the biosensor electrode and indicates that the reaction is surface-controlled and the direct electron transfer on the biosensor electrode takes place well [33, 34]. Hence, Mg doping could intensify the electrochemical response of TiO₂ nanodots based biosensor electrode.

(a)

(b)

3.3. Performance and Stability of Mg Doped TiO₂ Nanodots Based Biosensor Electrodes

Figure 6 shows the amperometric responses of the Nafion/HRP/Mg-TND/Ti biosensor electrodes with successive additions of 40 M H₂O₂. The sensitivity and detection limit of the electrodes were calculated and listed in Table 3; Nafion/HRP/Mg-TND-2/Ti electrode was demonstrated to be the best among them. For Nafion/HRP/Mg-TND-2/Ti biosensor electrode, the reduction current with addition of H₂O₂ increased rapidly and reached 95% of steady-state current within 3 s (Figure 6(a)). A linear relationship between current and H₂O₂ concentration was found in Figure 6(b), and the linear range of the current was from 6 M to 640 M with coefficient coloration (). Moreover, Nafion/HRP/Mg-TND-2/Ti electrode had a limit of detection (LOD) of 0.027 M (evaluated at a signal-to-noise ratio of 3, according to [35]) and the sensitivity of 1377.64 A mM⁻¹ cm⁻² which is 1.53-fold for the undoped electrode.

(a)

(b)

(c)

Although the sensitivities of the corresponding biosensor increased with the specific area of the nanodots films, the contribution of Mg ions is obvious. The specific area of Nafion/HRP/Mg-2-TND/Ti is just 1.09-fold of Nafion/HRP/TND-3/Ti electrodes (Table 2) but big difference in sensitivity was observed (Table 3). Hence, it is suggested that both of large specific area and proper Mg doping promote enzyme adsorption and function. The biosensor performance of Nafion/HRP/Mg-2-TND/Ti electrode is also better than those of HRP/TiO₂ microsphere/Nafion/Ti [27], Nafion/HRP/Au-TiO₂ nanoparticle/GCE [32], Nafion/HRP/TN-3/ITO [26], and HRP/Fe₃O₄/m-silica nanoparticle/SPE [29]. The stability of the Nafion/HRP/Mg-TND-2/Ti biosensor was investigated by amperometric technique, whereas the biosensor is stored for 12 days at 4°C. The current response of the biosensor was measured with the same conditions and it was found to be more than 89% retaining of the activity and indicates that the biosensor is stable for biosensor applications.

Moreover, the apparent Michaelis-Menten constant () was calculated from the Lineweaver-Burk equation because is one of the key measureable parameters related to working status of enzymes [36]: where is the steady-state current after the addition of substrate, is the maximum current measured under saturated bulk solution condition, and is the bulk concentration. The of Nafion/HRP/Mg-2-TND/Ti electrode was calculated to be 0.83 mM and to be smaller than Nafion/HRP/TND/Ti electrode (1.27 mM). This represents the fact that the enzyme achieves higher catalytic efficiency at low H₂O₂ concentration due to better affinity of the enzyme with Mg doped TiO₂.

4. Conclusions

Mg doped TiO₂ nanodots based electrode was prepared by sol-gel spin-coating, followed by calcination. It was found that 2% Mg doping TiO₂ nanodots electrode has better electrochemical response and biosensing performance than undoped electrode, with the sensitivity of 1377.64 A mM⁻¹ cm⁻², which is 1.53-fold. The reason is that Mg doping intensifies direct electrode transfer on the electrode due to good affinity of Mg doped TiO₂ with the working enzyme as well as increase of specific area of the nanodots. Such doping approach could provide an effective way to improve performance of the electrode of amperometric biosensors through chemical modifications.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work is financially supported by National Basic Research Program of China (973 Program, 2012CB933600), National Natural Science Foundation of China (51372217, 51272228), and Fundamental Research Funds for the Central Universities (2013QNA4010).

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Copyright © 2014 M. S. H. Al-Furjan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Advances in Materials Science and Engineering

Influences of Mg Doping on the Electrochemical Performance of TiO2 Nanodots Based Biosensor Electrodes

Abstract

1. Introduction

2. Experimental Design

2.1. Preparation and Characterization of Mg Doped TiO2 Nanodots Films on Ti Substrate

2.2. Preparation of H2O2 Biosensors

2.3. H2O2 Biosensors Characterization

3. Results and Discussion

3.1. Microstructures of Mg Doped TiO2 Nanodots Based Electrodes

3.2. Electrochemical Behaviors of Mg Doped TiO2 Nanodots Based Biosensor Electrodes

3.3. Performance and Stability of Mg Doped TiO2 Nanodots Based Biosensor Electrodes

4. Conclusions

Conflict of Interests

Acknowledgments

References

Copyright

Influences of Mg Doping on the Electrochemical Performance of TiO₂ Nanodots Based Biosensor Electrodes

2.1. Preparation and Characterization of Mg Doped TiO₂ Nanodots Films on Ti Substrate

2.2. Preparation of H₂O₂ Biosensors

2.3. H₂O₂ Biosensors Characterization

3.1. Microstructures of Mg Doped TiO₂ Nanodots Based Electrodes

3.2. Electrochemical Behaviors of Mg Doped TiO₂ Nanodots Based Biosensor Electrodes

3.3. Performance and Stability of Mg Doped TiO₂ Nanodots Based Biosensor Electrodes