Myoglobin- and Hydroxyapatite-Doped Carbon Nanofiber-Modified Electrodes for Electrochemistry and Electrocatalysis.

In this paper, a hydroxyapatite (HAp)-doped carbon nanofiber (CNF)-modified carbon ionic liquid electrode (CILE) was prepared and used for the investigation on the direct electrochemistry and electrocatalysis of myoglobin (Mb). HAp nanoparticles were mixed within a polyacrylonitrile (PAN) solution, and a HAp@PAN nanofiber was synthesized by electrospinning process, which was further controlled by carbonization at 800 °C for 2 h in a nitrogen atmosphere to get a HAp@CNF nanocomposite. Various techniques were used to check the physicochemical properties of HAp@CNF. Mb was mixed with a HAp@CNF dispersion solution and casted on the surface of CILE to obtain an electrochemical sensing platform. The direct electrochemistry of Mb on the modified electrode was checked when a pair of enhanced redox waves appeared, indicating the direct electron transfer of Mb. HAp@CNF exhibited high conductivity, good biocompatibility, and large surface area, which was beneficial for Mb immobilization. The modified electrode showed excellent electrocatalytic activity toward the reduction of trichloroacetic acid and sodium nitrite, which was further used to establish a new electroanalytical method. Real samples were analyzed by the proposed method with satisfactory results.

Introduction

Electrostatic spinning is a commonly used method that originated from Formalas’ patent and extended by Reneker’s researches,[1,2] which can be used for the rapid preparation of nanofibers with advantages such as being a simple device, low spinning cost, having a variety of spinnable materials, and controllable fiber scale.[3] Nanofibers that exhibit various structures and morphologies, including inorganic fiber, inorganic/organic hybrid fiber, and organic fiber, can be synthesized by this method.[4−6] As shown in Figure , an electrostatic spinning device is commonly composed of a high voltage supply, spinneret device, and target electrode.[7] By changing the experimental conditions such as the properties of the polymer solution, electric field intensity, and environmental conditions, electrostatic spinning technology is available to synthesize nanofibers with different diameters and densities,[8] which have been widely used in biomedicine for bone components, drug carriers, and sensing applications.[9−12] Also, the nanofiber manufactured from the organic polymers can be used for further fabrication of carbon nanofibers (CNFs) by high-temperature carbonization, which is a simple way for CNF preparation. For example, Kong et al. prepared a silicon nanoparticle-doped polyacrylonitrile (PAN) composite from electrostatic spinning and applied it to a lithium ion battery with good electrochemical properties.[13]

Figure 1

Schematic diagram of electrostatic spinning, high-temperature carbonization, electrode fabrication, and electrochemical application.

CNF is a kind of carbon nanomaterial with graphitic frameworks, high surface area, and specific conductive properties. Therefore, CNF has been widely used in various applications such as electrochemical biosensors, batteries, and catalyst supports.[14−16] Sun et al. prepared a nitrogen-doped CNF-modified carbon paste electrode (CPE) to determine bisphenol A.[17] Adabi et al. investigated the performance of electrodes that were modified with PAN-based CNFs.[18] Arkan et al. constructed an electrochemical sensor with TiO2 nanoparticles and electrospun CNF-modified CPE for the determination of idarubicin in biological samples.[19] Apetrei et al. prepared a gold nanoparticle/CNF-modified screen-printed carbon electrode to detect pyritinol in various samples.[20] Sakthivel et al. used a CoFeSe2 nanosphere and functionalized CNF composite-modified electrode for the electrochemical detection of caffeic acid.[21]

Hydroxyapatite (HAp) is an important component of the human skeleton and vertebrate hard tissue with excellent biocompatibility and biological activity,[22,23] which can be used in the medical fields for spinal fusion and bone defect treatment.[24,25] Also, HAp and its related composites can be used in electrochemical sensors. For example, El Mhammedi et al. proposed a HAp-modified CPE for para-nitrophenol detection.[26] Kanchana and Sekar reported the application of HAp as a sensing element for the electrochemical determination of folic acid.[27] Gao et al. utilized HAp and reduced graphene oxide or a carbon nanotube-modified electrode as an electrochemical sensor for the detection of hydrazine[28] and luteolin.[29] Pang et al. developed an electrochemical method for the determination of luteolin based on graphene and a HAp nanocomposite-modified glassy carbon electrode.[30] The combination of HAp with CNF can exhibit excellent synergistic effects and overcome some shortcomings such as the hydrophobicity of CNF and the low electron transfer ability of HAp. Wu et al. synthesized nanofibers by electrostatic spinning technology with HAp grown on the surface of CNFs.[31]

Myoglobin (Mb) is an oxygen heme protein that is mainly distributed in the myocardial and skeletal muscle tissue, which is composed of a single polypeptide and heme with the peptide chain coiled into a globular structure and heme packaged inside the cavitation. Heme is composed of ferroporphyrins that acted as an electroactive center in the redox reaction to lose and obtain electrons. Hence, Mb is commonly used as the model redox protein in the electrochemical sensor.[32−35]

In this paper, a HAp-doped PAN nanocloth was obtained by electrostatic spinning technology, which was carbonized at a high temperature to get a HAp-doped CNF (HAp@CNF). Then, HAp@CNF was mixed with the Mb solution and used as the modifier for the preparation of the electrochemical biosensor. By using a carbon ionic liquid electrode (CILE) as the substrate electrode, the modified electrode was named as Nafion/Mb-HAp@CNF/CILE and used as the working electrode in the electrochemical investigations. Direct electrochemical behaviors of Mb on the modified electrode were investigated by cyclic voltammetry when a pair of quasi-reversible redox peaks appeared, indicating that the existence of HAp@CNF accelerated the rate of electron transfer between Mb and a basal electrode. Then, the modified electrode was further used for the electrocatalytic reduction of trichloroacetic acid (TCA) and sodium nitrite (NaNO2).

Results and Discussion

Characterizations of the Materials

Figure displays scanning electron microscopy (SEM) images of CNF and HAp@CNF with different magnifications. It can be seen that the CNF exhibited a smooth surface with an average diameter of 220 nm, and HAp@CNF gave a rough fiber surface with irregular HAp clearly observed on the surface of CNF. Transmission electron microscopy (TEM) images of HAp@CNF proved that HAp was distributed homogeneously in CNF (Figure e,f). X-ray photoelectron spectroscopy (XPS) results can be used to characterize the chemical structure and elemental composition on the surface of HAp@CNF. As shown in Figure g, the peaks of CNF were attributed to C1s, N1s, and O1s. The spectrum of HAp@CNF with new peaks of Ca2s, Ca2p, P2s, and P2p indicated that HAp crystals were successfully doped within the CNF.[31] The Raman spectrum gave two peaks at 1326.8 and 1583.5 cm–1 (Figure h), which were attributed to two characteristic bands of D and G bands, respectively. The D band was attributed to the vibration of carbon atoms in disordered carbon structures, whereas the G band was related to the in-plane vibration of sp2 carbon atoms. The intensity ratio of ID/IG was calculated to be 1.06, which indicated a high-level disorder of HAp@CNF.[36,37]

Figure 2

SEM images of (a, b) CNF and (c, d) HAp@CNF at various magnifications. (e, f) TEM images of HAp@CNF. (g) XPS spectra of HAp@CNF and CNF. (h) Raman spectrum of HAp@CNF.

Spectroscopic results of Mb and Mb-HAp@CNF were further checked with the data shown in Figure S1. The peak positions remained unchanged in Fourier transform infrared (FT-IR) and UV–vis spectroscopy, which proved that Mb remained its natural structures after mixing with HAp@CNF due to its biocompatibility.

Direct Electrochemical Behavior of the Mb-Modified Electrodes

Figure a–d shows the cyclic voltammetric responses of Nafion/CILE, Nafion/HAp@CNF/CILE, Nafion/Mb/CILE, and Nafion/Mb-HAp@CNF/ CILE, respectively, in phosphate-buffered saline (PBS) (pH 3.0) at a scan rate of 100 mV s–1. On Nafion/CILE and Nafion/HAp@CNF/CILE, no electrochemical responses were observed, which indicated that no electrochemical reaction took place. On Nafion/Mb/CILE, a pair of redox couples with a deformed shape indicated a slow electron transfer rate. With the further incorporation of HAp@CNF on the modified electrode, a pair of well-defined quasi-reversible redox peaks appeared with a formal peak potential (E0′) of −0.196 V and a peak-to-peak separation (ΔEp) of 66 mV. Therefore, the presence of the HAp@CNF nanocomposite on the electrode surface could improve the electron transfer of Mb with the underlying electrode. CNF has been proven to be an effective conductor for electron transfer due to its carbon nature with long-distance electron communication ability. HAp is a biocompatible material for Mb to retain the biostructure without deformation. Also, the presence of HAp in CNF can modulate the interfacial hydrophilicity of the composite, which enables the easy interaction of Mb with HAp@CNF. The interfacial information of the modified electrode can be tested by electrochemical impedance spectroscopy (EIS) with the data shown in Figure S2. The Ret values of Nafion/HAp@CNF/CILE, Nafion/CILE, Nafion/Mb-HAp@CNF/CILE, and Nafion/Mb/CILE were calculated to be 19.8, 46.7, 77.1, and 110.2 Ω, respectively, indicating that the presence of HAp@CNF was beneficial for the electron transfer with the decrease in the interfacial resistance.

Figure 3

Cyclic voltammograms of (a) Nafion/CILE, (b) Nafion/HAp@CNF/CILE, (c) Nafion/Mb/CILE, and (d) Nafion/Mb-HAp@CNF/CILE in pH 3.0 PBS at a scan rate of 100 mV s–1.

Electrochemical Investigations

The influence of the scan rate on the electrochemical behaviors of Nafion/Mb-HAp@CNF/CILE was studied by cyclic voltammetry with the results shown in Figure A. The redox peak currents varied linearly with scan rate in the range of 0.05–1.0 V s–1, and the linear regression relationships were shown in Figure B, indicating a thin-layer surface-controlled electrochemical behavior. The redox peak potentials were also shifted gradually with the increase of peak-to-peak separation (ΔEp), and Figure C shows the linear regression relationships of Ep with ln υ. According to Laviron’s equations,[38] the values of the electron transfer number (n), the electron transfer coefficient (α), and the heterogeneous electron transfer rate constant (ks) were estimated to be 0.95, 0.45, and 1.10 s–1, respectively. The obtained ks value was larger than some of the reported values for different Mb-modified electrodes with the compared results shown in Table , which indicated that HAp@CNF on CILE formed a conductive network for Mb with a faster electron transfer rate. The effect of buffer pH on the direct electrochemical behavior of Nafion/Mb-HAp@CNF/CILE was further investigated in PBS with a pH range of 3.0–8.0. As shown in Figure D, the redox peak potential of Mb moved to a negative direction, indicating that the proton was involved in electrode reaction. E0′ had a good linear relationship with buffer pH, and the linear regression equation was obtained as E0′ (V) = −0.0541pH + 0.016 (n = 6, γ = 0.994). The slope value of −54.1 mV pH –1 was close to the theoretical value of the Nernst equation (−59.0 mV pH –1) for a one-electron and one-proton transfer reversible electrode reaction. Furthermore, the biggest peak current was observed at pH 3.0, which was chosen as a supporting electrolyte for electrochemical investigation, and a sufficient amount of protons were present at this condition.

Figure 4

(A) Influence of scan rate on the electrochemical responses of Nafion/Mb-HAp@CNF/CILE in pH 3.0 buffer with different scan rates (from (a)–(k), 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mV s–1, respectively). (B) Linear relationship of the redox peak currents versus scan rate (υ). (C) Linear relationship of the redox peak potentials versus ln υ. (D) Cyclic voltammogram of Nafion/Mb-HAp@CNF/CILE in different pH PBS values (from (a)–(f), 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively) with a scan rate of 100 mV s–1.

Table 1

Comparison of ks with Various Mb-Modified Electrodes

different electrodes	pH	ks (s–1)	refs
Mb-HSG-SN-CNTs/GCE	5.0	0.410	(39)
Nafion/Mb-GR-Pt/CILE	7.0	0.584	(40)
Mb-ZnO-modified GE	7.0	1.000	(41)
Nafion/Mb/MWCNTs/CILE	7.0	0.332	(42)
Mb/NiO NPs/GCE	7.0	0.340	(43)
CTS/Mb/SWCNHs/CILE	3.0	0.590	(44)
Nafion/Mb-HAp@CNF/CILE	3.0	1.100	this work

Electrocatalytic Activity

The elecrocatalytic activity of Nafion/Mb-HAp@CNF/CILE to TCA and NaNO2 was investigated by cyclic voltammetry with the results shown in Figure . As shown in Figure A, the addition of different concentrations of TCA resulted in the increase in the reduction peak current when the oxidation peak disappeared and the second reduction peak appeared simultaneously at −0.528 V, indicating a typical electrocatalytic behavior of the redox protein-modified electrode.[45] A good linear relationship of TCA concentration with a reduction peak current (Ipc) was obtained (inset of Figure A) in the range of 6.0–180.0 mM. The linear regression equation was Ipc (μA) = 6.82C (mM) + 33.02 (n = 22, γ = 0.998) with the limit of detection (LOD) of 2.0 mM (3σ). When the concentration of TCA was more than 180.0 mM, the peak current reached to a platform, which was the characteristic of Michaelis–Menten kinetics performance. The apparent Michaelis–Menten constant (KMapp) could be calculated to be 0.224 M, which was due to the presence of the CNF network with an excellent electrical conductivity and large specific surface area that increased the load amount of Mb molecules on the electrode surface.

Figure 5

Cyclic voltammograms of Nafion/Mb-HAp@CNF/CILE in pH 3.0 buffer with (A) TCA (6.0, 70.0, 80.0, 90.0, 100.0, 110.0, 120.0, 130.0, 140.0, 150.0, 160.0, 170.0, and 180.0 mM; inset shows the linear relationship of catalytic peak currents and TCA concentration) and (B) NaNO2 (0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.2, 1.6, 2.0, 3.0, 4.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 10.0 mM; inset shows the linear relationship of catalytic peak currents and the NaNO2 concentration); scan rate, 100 mV s–1.

The elecrocatalytic activity of Nafion/Mb-HAp@CNF/CILE with different concentrations of NaNO2 was checked with the typical cyclic voltammograms shown in Figure B. A new reduction peak was found at −0.798 V, and the reduction peak current increased with the NaNO2 concentration in two sections (0.3–1.6 and 2.0–10.0 mM) with two linear regression equations: Ip (μA) = 50.1C (mM) + 17.4 (n = 9, γ = 0.992) and Ip (μA) = 3.93C (mM) + 99.0 (n = 10, γ = 0.995). The LOD was calculated to be 0.23 mM (3σ), and the KMapp was estimated to be 1.13 mM.

A comparison of this method with other reported results is summarized and compared in Table for TCA and Table for NaNO2, which indicated a relatively wider linear range or lower detection limit for the detection of the target analytes.

Table 2

Comparison of Analytical Performances of Different Electrodes for TCA

electrodes	linear range (mM)	LOD (mM)	refs
Nafion/Mb-Co3O4-Au/IL-CPE	2.0–20.0	0.5	(46)
Nafion/Mb/NiO/GR/CILE	0.69–30.0	0.23	(47)
{PDDA/Hb}8/PGE	3.92–58.4	1.98	(48)
CTS/ELDH-GR-Hb/CILE	5.0–135.0	1.506	(49)
CTS-Mb-GR-IL/CILE	2.0–16.0	0.583	(50)
Nafion/Mb-HAp@CNF/CILE	6.0–180.0	2.0	this work

Table 3

Comparison of Analytical Performances of Different Electrodes for NaNO2

electrodes	linear range (μM)	LOD (μM)	refs
GH-CS/Fc-NH2/Cytc/GCE	0.1–150	0.04	(51)
CR-GO/GCE	8.9–167	1.0	(52)
Hb/Au/GCE	4–350	1.2	(53)
CTS/TiO2-Hb/CILE	800–20000	260	(54)
Nafion-Mb-SGO-GCE	2.0–24.5	1.5	(55)
Nafion/Mb-HAp@CNF/CILE	0.3–10.0	0.23	this work

Analytical Application

In order to verify the real application of this electrochemical sensor, the TCA content in the medical facial peel sample was checked by the standard addition method. A 10 μL medical facial peel solution (35%) was diluted by 0.1 M PBS (pH 3.0) to a final 10 mL volume to get the sample solution, which was put into an electrochemical cell and detected by the three-electrode system. As shown in Table , the recovery was calculated from 94.7 to 106.0%, indicating the practical application in drug sample detection.

Table 4

Detection Results of Medical Facial Peel Samples by This Method (n = 3)

sample	detected (mM)	added (mM)	total (mM)	recovery (%)
medical facial peel solution	15.8	10.0	25.4	96.0
		20.0	37.4	106.0
		30.0	44.2	94.7

Conclusions

HAp@CNF was synthesized by an electrostatic spinning HAp@PAN solution with the following high-temperature carbonization process, which was used for the electrode modification with direct electrochemistry of Mb realized. The fabricated Nafion/Mb-HAp@CNF/CILE showed a pair of well-defined redox peaks on cyclic voltammograms with an enhanced electrochemical response, indicating that the HAp@CNF nanocomposite was beneficial to enhance the electrochemical signal of Mb on the electrode. The modified electrode showed excellent electrocatalytic reductions for TCA and NaNO2 with a wide linear response and high sensitivity, which could also be applied to detect the TCA content in the medical facial peel solution.

Experimental Section

Reagents

PAN (MW 150,000; J&K Chem. Co., China), N,N-dimethylformamide (DMF; Xilong Chem. Co., China), Mb (MW, 17,800, Sigma-Aldrich Co., USA), 1-hexylpyridinium hexafluorophosphate (HPPF6; Lanzhou Yulu Fine Chem. Ltd. Co., China), graphite powder (particle size, 30 μm; Shanghai Colloid Chem. Co., China), TCA (Tianjin Kemiou Chem. Ltd. Co., China), NaNO2 (Yantai Sahe Chem. Ltd. Co., China), and medical facial peel (35%; Shanghai EKEAR Biotech. Ltd. Co., China) were used as received. HAp was synthesized based on the reported procedure.[56] The supporting electrolyte was 0.1 M phosphate-buffered saline (PBS), which was deoxygenated with pure nitrogen before the experiments. All the other chemicals were of analytical grade, and the solutions were prepared by using double distilled water.

Instruments

Voltammetry and electrochemical impedance spectroscopy (EIS) were performed with CHI 1210A and CHI 660D electrochemical workstations (Shanghai CH Instrument, China), respectively. A three-electrode system was used for the electrochemical experiments, which was composed of the working electrode (Nafion/Mb-HAp@CNF/CILE), the auxiliary electrode (platinum wire electrode), and the reference electrode (saturated calomel electrode). Scanning electron microscopy (SEM) was performed by a JSM-7100F scanning electron microscope (JEOL, Japan) with transmission electron microscopy (TEM) on a JEM 2010F instrument (JEOL, Japan). Raman spectrum was obtained on a LabRAM HR system using 532 nm lasers (Horiba, France) with X-ray photoelectron spectroscopy (XPS) on an AXIS HIS 165 spectrometer (Kratos Analytical, UK). FT-IR was carried out using a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific Inc., USA). UV–vis spectroscopy was recorded on a TU-1901 double-beam UV–vis spectrophotometer (Beijing General Instrument Ltd. Co., China). The carbonization process was performed by a BTF-1200C vacuum tube furnace (Anhui Best Equipment Technology Ltd. Co., China).

Synthesis of the HAp@CNF Nanocomposite

HAp@CNF was synthesized by electrospinning the PAN and HAp mixture with the following carbonization. In general, 0.4 g of HAp was dispersed into 10 mL of DMF with the addition of 1.6 g of PAN, which was stirred completely to form a pale yellow colloidal solution. The parameters for electrostatic spinning were set as follows: the voltage of 8.80 kV, the flow rate of jet spinning of 12 μL min–1, the distance of the needle to the receiver of 12 cm, and the cylinder receiver rotated with a speed of 940 rpm. The resulted HAp-doped PAN nanocloth was placed in a tube furnace and heated at 800 °C for 2 h in a nitrogen atmosphere. Finally, the HAp@CNF nanocomposite could be obtained after cooling down to room temperature in a nitrogen atmosphere.

Preparation of Nafion/Mb-HAp@CNF/CILE

CILE was manufactured as a basal electrode with 0.8 g of HPPF6 and 1.6 g of graphite powder, which were mixed and ground carefully to form a homogeneous carbon paste. Then, it was packed into a glass tube electrode (Φ = 4.0 mm), and the electrical contact was established through a copper wire at the end of the paste. The CILE surface was polished on a weighing paper before use.

HAp@CNF was ground in a mortar to a black powder, and a 3.0 mg mL–1 HAp@CNF dispersion solution was prepared by ultrasound and oscillation, which was mixed with a 30 mg mL–1 Mb solution and shocked to a homogeneous mixture. Then, 8 μL of Mb-HAp@CNF dispersion was casted on the surface of CILE and dried at room temperature to get the modified electrode (Mb-HAp@CNF/CILE). Finally, 6 μL of 0.5% Nafion solution was spread on Mb-HAp@CNF/CILE to get the modified electrode (Nafion/Mb-HAp@CNF/CILE).

Analytical Procedure

Electrochemical measurement was operated in 0.1 M pH 3.0 PBS that was deoxygenated by purified nitrogen for 30 min before the experiments. UV–vis absorption spectroscopic experiments were performed with a mixture solution composed of Mb and HAp@CNF with the wavelength scanned from 300 to 600 nm. The film of the Mb solution and Mb-HAp@CNF solution dried on a glass slide was scraped for FT-IR experiments.