The results from the previous section showed the utility of MWCNT-Ti as an electrode for detecting Fe2+/3+ redox couples. For biological applications, it is also necessary to show that MWCNT-Ti can detect redox reactions associated with osteoblast differentiation. The extracellular components from osteoblasts in an HCl solution have been used to mimic the biological environment around an orthopedic implant. Figure 8a, b show that the bare Ti and anodized Ti electrodes cannot detect any redox process. In contrast, the results in Fig. 8c confirmed that MWCNTs enhanced the direct electron transfer through Ti by adding a highly conductive surface the MWCNTs.

After decreasing the surface area of the MWCNT-Ti electrode from 1 cm2 to 1 mm2, the redox potentials also decreased as shown in Figs. 8c and 9b. The faradic current of the oxidation process dropped approximately ten times with respect to the decrease in surface area A, corresponding to equation (1). Corresponding with the Randles-Sevcik equation, the peak current was linearly proportional to the area. When plotting the anodic (/ ) and cathodic peak (/ ) currents of MWCNT-Ti, a linear relationship to the square root of the scan rates was observed, as shown in Fig. 9c.

The CV showed well-defined redox peaks in the osteoblast supernatants at all concentrations. The peaks detected in the solution for the calcium concentrations of 1.481 mg/cm2 (after 7 days), and 1.597 mg/cm2 (after 14 days) have less faradic current and I-V graph area than the concentration of 2.48 mg/cm2 (after 21 days), as shown in Fig. 10a, b. Typically, the more HA deposited and synthesized by osteoblasts, the higher the calcium and protein concentrations. Therefore, it is likely that the supernatant from 21 days had higher protein concentrations than the solutions from 7 and 14 days, leading to the stronger redox reactions and higher capacitance between the electrode surfaces.

In addition, interpretation of CV results must consider other factors. In particular, a measurement of the redox reactions when oxygen was dissolved in the electrolyte solution showed that the peak currents were shifted toward the negative. Furthermore, a change in the pH of the electrolyte solution and the presence of

Potential (mV) vs. Ag/AgCl Potential (mV) vs. Ag/AgCl

Potential (mV) vs. Ag/AgCl Potential (mV) vs. Ag/AgCl

Fig. 8 Cyclic voltammograms with an electrolyte solution of the extracellular matrix secreted by osteoblasts after 21 days of culture for: (a) conventional Ti; (b) anodized Ti; and (c) MWCNT-Ti with a working area of 1 cm2. (d) CV of all three electrodes in comparison. Only MWCNT-Ti possessed the quasireversible redox potential, while conventional Ti and anodized Ti did not

Potential (mV) vs. Ag/AgCl Potential (mV) vs. Ag/AgCl

Fig. 8 Cyclic voltammograms with an electrolyte solution of the extracellular matrix secreted by osteoblasts after 21 days of culture for: (a) conventional Ti; (b) anodized Ti; and (c) MWCNT-Ti with a working area of 1 cm2. (d) CV of all three electrodes in comparison. Only MWCNT-Ti possessed the quasireversible redox potential, while conventional Ti and anodized Ti did not water may also shift the current and affect the potential of the redox peaks. Thus, CV with the potential range between -1 and 1 V on MWCNT-Ti was performed in a pure solution of 0.6 N HCl without other proteins or ions. An H+ reduction peak at a potential between -0.5 and -0.8 V was found (data not shown). Moreover, the H+ reduction peak still appeared when window ranges from -5 to 5 V and from -0.35 to 0 V were used, confirming the H2O decomposition occurred in our CV experiments.

It is clear that the MWCNT-Ti electrodes showed a potential for better performance than commercially available electrodes, such as GCE and PTE. When using the sweep voltage from -1 to 1 V, a set of mixed redox peaks with two oxidations and two reductions appeared (data not shown). These two redox peaks in the CV, indicated



















250 mV/s .¿py

150 mV/s


— 200 mV/s

300 mV/s

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-300 -250 -200 -150 -100 Potential (mv) vs. Ag/AgCl

-300 -250 -200 -150 -100 Potential (mv) vs. Ag/AgCl

y = 0.152x - 0.0072

R2 = 0.9787

1 0.4 0.6


y = -0.1954x - 0.0046

R2 = 0.9915

■ Cathodic (Ipc) ♦ Anodic (Ipa)

-300 -250 -200 -150 -100 Potential (mV) vs. Ag/AgCl

-300 -250 -200 -150 -100 Potential (mV) vs. Ag/AgCl

Fig. 9 Cyclic voltammograms with an electrolyte solution of the extracellular matrix secreted by osteoblasts after 21 days of culture for (a) conventional Ti and (b) MWCNT-Ti with a working area of 1 mm2. (c) Plot of the experimental cathodic and anodic peak currents, obtained from (b), versus the square root of the scan rates; and (d) the compared capacitance of MWCNT-Ti and Ti

that the process likely involved more than one electron transfer. For each couple, the amplitude and position of the oxidation peak in CV was unequal to those of the reduction peak. The second oxidation peak (omitted from Figs. 8c or 9b) was observed at 236 mV for the MWCNT-Ti electrode, 405 mV for the GCE, and -33 mV for the PTE, all with the scan rate of 100 mV/s. However, for the GCE and the PTE electrodes, a second reduction did not appear, while the MWCNT-Ti showed two complete and well-defined redox reactions. These two peaks reflected that more than one electron transfer was involved, since one occured at a positive potential and one at a negative in the CV. Further, the current amplitude for the MWCNT-Ti peaks was noticeably larger than that for GCE and PTE. Thus, it can be concluded that the MWCNT-Ti electrode has higher electron transfer as well.





7 Days

14 Days 21 Days

Fig. 10 (a) Cyclic voltammograms of MWCNT-Ti electrodes in an electrolyte solution of the extracellular matrix secreted by osteoblasts cultured on conventional Ti after 21 days. (b) Results from the calcium deposition assay determined the calcium concentrations in an electrolyte solution of the extracellular matrix secreted by osteoblasts on conventional Ti after 7, 14, and 21 days of culture. Data=mean ± S.E.M; n = 3; *p < 0.01 (compared to 7 and 14 days)

At first it can be hypothesized that these redox reactions happened due to the redox of inorganics (such as Ca or P). In the case of Ca, the reduction of Ca2+ to Ca crystals (nucleation) typically is expected with the reduction current for a highly negative potential. A positive feedback IR compensation (IRC) needs to be applied to the CV in order to drive the oxidation current to dissolve the Ca crystal into Ca2+. For example, an overpotential of more than -1 V, IRC of 0.771 W, melting temperature of 900°C, and scan rate of 50 mV/S were required (Chen and Fray 2002). Without applying IRC in this study, the diffusion-controlled behavior, which elec-trochemically depletes calcium cations near the electrode surface, has been impossible to achieve. In addition, the reduction and oxidation of titanium oxides, which are the reaction counterparts of calcium redox, appeared at a positive potential between 0 and 0.25 V, respectively, which did not appeared in our CV studies. In the case of P, the cyclic voltammetry experiments were performed further in phosphate buffered saline (PBS). A final concentration of 1 M PBS, which was prepared in-house, had 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.4. The metal orthophosphate ions exhibited a high stability to reduce from P(V) (such as PO4-, PO-) to P(III), or further to P(I). However, no redox peaks appeared in the potential range of -1 to 1 V (data not shown). It is therefore likely that neither the redox reactions of calcium ions nor phosphate ions occurred due to the absence of the peaks. Instead, it has been hypothesized that the appearance of the redox peaks when using an MWCNT-Ti electrode is likely because of the reduction and oxidation of proteins from the osteoblast supernatant.

The observed quasireversible redox reaction reflected the fact that MWCNT-Ti sensors promoted electrochemical reactions. However, an irreversible process can occur at the interface of an electrode due to the denaturation of proteins, or ion reduction. Typically, when the carbon bonds of proteins are altered, only irreversible organic reduction or oxidation happens (Schuring 2000). While the configuration of ions (Ca2+, PO4-, OH-), which remain unchanged, also can lead to the irreversible process due to only its reduction or oxidation. Importantly, without MWCNTs on the Ti surface, nonspecific protein adsorption may readily occur, leading to an electro-chemically insulating surface layer. Nevertheless, the redox on MWCNT-Ti did not decrease its oxidation and reduction current after applying the sweep voltage for some time, so protein adsorption did not occur apparently in significant amounts at the MWCNT-Ti electrode. Indeed, in the osteoblast supernatant, the MWCNT-Ti electrode provides a relatively specific surface to which proteins can bind reversibly by diffusion while retaining their function. The protein redox is also dependent on the pH of the electrolyte solution (acidic or basic) (Battistuzzi et al. 1999). This shift appears due to the influence of 0.6 N HCl with H2O as a solvent which influences proteins, leading the peak shift into the negative potential, as shown in Figs. 8c, 9b, and 10a.

It has been hypothesized that proteins were involved in these redox reactions at the surface of MWCNT-Ti in the solution of osteoblast extracelluar components. If those proteins maintain their function and are detected by the MWCNT-Ti electrode, the excellent electrochemical behavior explained here may show that MWCNT-Ti sensors are superior candidates for biosensor applications. However, investigating the type and size of acidic resistant proteins is still in progress.

As shown here, the electronic performance of MWCNT-Ti sensors, but not the bare Ti, provides excellent redox reactions. As mentioned, the electrochemical experiments were performed so far without degassing with bubble nitrogen gas. This omission may have resulted in an apparent background signal, which was mixed and appeared in current-voltage curves (CV). Moreover, the redox reaction at the MWCNT-Ti sensor mentioned here may depend on various ion sources, including reversible protein adsorption, dissolved oxygen, and the acidic nature of HCl. However, the redox reactions of iron (II/III) and the osteoblast extracellular components still occurred only when using MWCNT-Ti sensors, and not when using Ti or anodized Ti. Importantly, it was shown that MWCNT sensors performed as the electrocatalysts for the oxidation and reduction of iron (II/III) and of extracellular components, which included various proteins and inorganic substances synthesized and deposited by osteoblasts.

The electrolyte solution of osteoblast extracellular components was composed of inorganic and organic substances. This solution was not classified in terms of the types of molecules in solution, but rather, all components were dissolved together in the acidic solution and used in all cyclic voltammetry experiments in this study. Future work needs to classify the type of proteins in such osteoblast supernatants.

However, it is likely that in vivo the capacitance and the electroactive surface of Ti will be enhanced after bone tissue is formed without the presence of MWCNTs. After hip implant insertion, blood and body fluids surround the Ti implant, creating a specific capacitance for the Ti electrode in the beginning. Within 1 month, as osteoblasts deposit calcium around the implant, the capacitance of the implant and bone tissue increases. Cell membranes also have electric potentials and perform redox process (Jeansonne et al. 1978, 1979), and it has been found that after 14 days in vitro these redox processes at the surface of bare Ti increased.

In vitro, nevertheless, MWCNTs have been used to modify the electrodes to detect other biomolecules and enhance their redox reactions, such as NADH (Lin et al. 2005), hydrogen peroxide (Kurusu et al. 2006; Lin et al. 2005), glucose (Liu et al. 2005), putrescine (Luong et al. 2005), and DNA (He et al. 2006). Specifically, the growth of MWCNTs from nanoporous metals electrodes can enhance many diverse sensor applications. For example, MWCNTs functionalized with thiol groups have been used for sensing aliphatic hydrocarbons (such as methanol and ethanol), forming unique electrical identifiers (Padigi et al. 2007). Moreover, MWCNTs grown on silicon substrates enhanced ionic conductivity and have been integrated with unmodified plant cellulose as a film, and used as a cathode electrode in a lithium ion battery, or as a supercapacitor with bioelectrolytes (such as biological fluid and blood) (Pushparaj et al. 2007). Interestingly, both supercapacitors and batteries derived from a MWCNTs-nanocomposite film, can be integrated to build a hybrid, or dual-storage battery-in-supercapacitor device. The cytocompatible MWCNTs-device may be useful to integrate with an orthopedic-implant biosensor as passive energy storage.

Not only can an implanted electronic circuit be powered by an implant battery (fabricated or self-integrated) within the implant material, but it also can be induced by an external power supply. For orthopedic applications, telemetric devices started in 1966 and became imperative as a telemetric orthopedic implant in order to transmit a signal to an external device (Kaufman et al. 1996; Bergmann et al. 2001a, b; D'Lima et al. 2005; Graichen and Bergmann 1991; Graichen et al. 1999). For example, Graichen et al. (2007) developed a new 9-channel telemetry transmitter used for in vivo load measurements in three patients with shoulder Ti endoprosthesis. The low radio frequency (RF) of an external inductive power source generates power transmission through the implanted metal. At the end of the Ti implant, the pacemaker feedthrough rounds a single loop antenna to transmit the pulse interval signal, which is modulated at a higher RF by the microcontroller, to an external device. Then, the RF receiver of the external device synchronizes with the modulated pulse interval to recover the data stream and report to a clinician. Thus, either an implanted battery or a telemetry system can supply the electrical power to a calcium-detectable chip inside the orthopedic implant for clinical use.

The automatically-sensing concept for bone growth juxtaposed to orthopedic implants can also be applied for determining infection and scar tissue formation. An energy source energizes a telemetric-implanted circuit, and allows it to transmit data to an external receiver to determine bone growth. Indeed, the electrochemical biosensor may reduce the complexity of imaging techniques and patient difficulties.

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