Laser-scribed Graphene Electrodes Functionalized with Nafion/Fe3O4 Nanohybrids for the Ultrasensitive Detection of Neurotoxin Drug Clioquinol.

The analysis of pharmaceutical active ingredients plays an important role in quality control and clinical trials because they have a significant physiological effect on the human body even at low concentrations. Herein, a flexible three-electrode system using laser-scribed graphene (LSG) technology, which consists of Nafion/Fe3O4 nanohybrids immobilized on LSG as the working electrode and LSG counter and reference electrodes on a single polyimide film, is presented. A Nafion/Fe3O4/LSG electrode is constructed by drop coating a solution of Nafion/Fe3O4, which is electrostatically self-assembled between positively charged Fe3O4 and negatively charged Nafion on the LSG electrode and is used for the first time to determine a neurotoxicity drug (clioquinol; CQL) in biological samples. Owing to their porous 3D structure, an enriched surface area at the active edges and polar groups (OH, COOH, and -SO3H) in Nafion/Fe3O4/LSG electrodes resulted in excellent wettability to facilitate electrolyte diffusion, which gave ∼twofold enhancement in electrocatalytic activity over LSG electrodes. The experimental parameters affecting the analytical performance were investigated. The quantification of clioquinol on the Nafion/Fe3O4/LSG electrode surface was examined using differential pulse voltammetry and chronoamperometry techniques. The fabricated sensor displays preferable sensitivity (17.4 μA μM-1 cm-2), a wide linear range (1 nM to 100 μM), a very low detection limit (0.73 nM), and acceptable selectivity toward quantitative analysis of CQL. Furthermore, the reliability of the sensor was checked by CQL detection in spiked human blood serum and urine samples, and satisfactory recoveries were obtained.

Introduction

In recent years, the analysis of drugs and their active ingredients has become an integral part of drug quality control and clinical trials. Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQL) is a halogenated analogue of 8-hydroxyquinoline used to treat gastrointestinal amebiasis and skin illness.[1] It can also serve as a Zn2+ and Cu2+ chelator, and when combined with cobalamin, it can treat Parkinson’s and Alzheimer’s disorders.[2] An overdose or prolonged administration of the CQL drug may cause subacute myelo-optic neuropathy (SMON), which is a relatively uncommon neurological disorder. The SMON syndrome is an iatrogenic disease in which nerve fibers in the spinal cord and optic nerves degenerate. It was most prevalent in Japan. Several symptoms are associated with SMON, including sensory losses, dysesthesia, ataxic paralysis, impaired vision, and so forth.[3,4] Thus, the use of CQL has been banned in Japan and some other countries. Despite this, CQL is widely used as a therapeutic agent in several countries. Thus, developing simple, rapid, and trustworthy protocols for monitoring and detecting CQL is imperative.

Several approaches have been developed to analyze CQL, including gravimetry,[5] titrimetry,[6] calorimetry,[7] chromatography,[8] spectrophotometry,[9] and fluorescence detection.[1,10] Despite their advantages of sensitivity and accuracy, these techniques are not easy in practical analysis because they are very expensive to operate, complex, employ costly instruments, and involve lengthy sample preparation procedures and complicated manipulations. In this concern, electroanalytical methods are highly efficient, straightforward, sensitive, and selective. As far as we know, most of the research is devoted to develop high-performance electrode materials or improve the properties of electrochemical sensors and catalytic performances.[3,4,11−15] Nevertheless, to test at point-of-care (POC), portable, reusable, and miniaturized CQL sensors are still indispensable.

Graphene and graphitic materials are ideal candidates for electrochemical sensors because they possess a wide electrochemical potential window and demonstrate electro-catalytic activity in a variety of redox reactions.[16−20] Fabrication of graphene-based materials often relies on chemical vapor deposition, self-reduction of graphene oxide, or other approaches that are usually pricey and labor-intensive.[21−23] In contrast, graphene electrodes, however, can be imprinted using printing techniques (e.g., inkjet printing and screen printing) using graphene flakes made from chemically exfoliating bulk graphitic materials.[24−27] However, these printing techniques often require additional post-printing processes (e.g., laser, thermal, or photonic annealing) to make the graphene electrodes more electrically conductive, which further complicates their fabrication.[19,28,29] Furthermore, solution processing of graphene could result in restacking and reduced surface areas.[30] Therefore, implementation of a new approach may ultimately provide a sustainable alternative for mass production of graphene with low toxicity and a favorable environment for biomolecules. Laser-scribed graphene (LSG) is an efficient and scalable technique for generating conductive 3D porous graphene through laser etching of thermoplastic resins such as polyimide (PI).[31−33] Currently, conventional substrate electrodes like glassy carbon/metal electrodes are being replaced with LSG electrodes because of their flexibility, large active surface area, good conductivity, ease of manufacture, and low cost.[23,34,35] Considering all these features, LSG electrodes show great potential as disposable and portable biosensor strips.

To date, much attention has been focused on using metal/metal oxide nanoparticles (NPs) on carbon-based materials as a signal transducing and sensing material to improve biosensing capability.[36,37] The incorporation of NPs into LSGs has resulted in hybrid materials that exhibit improved physical and chemical properties. A number of metal oxides, such as CO3O4, MoO2, and Fe3O4, can be grown on the surface of laser-induced graphene (LIG), demonstrating the versatility of this method for synthesizing metal oxide–LIG hybrid nanostructures.[38] Among the metal oxide NPs decorating LIG, Fe3O4 composed of Fe2+ and Fe3+ has been widely recognized as an effective electrocatalyst due to its excellent electronic conductivity, exceptional catalytic activity, rapid electron transfer ability, low cost, simplicity in synthesis, biocompatibility, chemical stability, and ease of functionalization.[39] Nanohybridization is considered to be a convenient method of preparing hybrid networks with desirable physical properties by incorporating inorganic polymers into carbon nanomaterials while preserving the intrinsic characteristics of carbon.[40] The ionic polymer Nafion has been widely utilized for the preparation and modification of different electrochemical sensors.[41,42] It is well known that Nafion comprises mainly hydrophobic backbone chains (−CF2 groups) and hydrophilic side chains (−SO3– groups), which can functionalize graphene in alcoholic solutions after it is added to graphene.[43] Furthermore, sulfonic groups prevent graphene layers from stacking.[44] A hybrid nanostructure made by combining Fe3O4 NPs, Nafion, and graphene-based nanomaterials accelerates electron transfer processes at the electrode/electrolyte interface, making them suitable for use in electrochemical biosensors. As a result, Fe3O4 combined with LSG and Nafion is expected to produce a remarkably effective electrocatalyst for the sensitive determination of therapeutic agents. Recently, electrochemical sensors based on LSG have been successfully applied for the detection of small biomolecules, potential biomarkers, and neurotransmitters.[31,45−52] Nasraoui et al. described the fabrication of LIG modified with functionalized multiwalled carbon nanotubes/AuNPs for nitrite detection in water samples.[53] Zhang et al. developed LIG decorated with Cu NPs using electroless deposition technique to fabricated glucose sensor strips to detect low glucose levels in the serum samples.[54] Although, LIG electrode patterns with bimetals (Ni/Au) have been leveraged for non-enzymatic glucose sensors.[55] Poly(3, 4-ethylenedioxythiophene) was electrochemically deposited on LSG to fabricate flexible electrochemical sensors for the simultaneous detection of DA, AA, and UA.[56] You’s group synthesized Nobel metal NPs–LIG hybrid nanostructures using laser induction, which were later used to fabricate a flexible immunosensor to detect Escherichia coli O157:H7.[38] In addition, the operation of supercapacitors on Fe3O4 NP-anchored LIG (LIG/Fe3O4) electrodes was studied.[57−59] To our knowledge, no study has reported the detection of CQL using LSGs modified with Nafion/Fe3O4.

In line with these perspectives, we proposed a Nafion/Fe3O4 nanostructured anchored LSG-based electrochemical sensor strip for the detection of the neurotoxic drug clioquinol. To characterize the physicochemical properties of these electrodes, various techniques (analytical and spectroscopic) were used. The homogenous distribution of Fe3O4 coupled with the high electrostatic interaction of Nafion–LSG in a super-hydrophobic environment provides a high surface-to-volume ratio that makes electrolyte access to the electrode surfaces easier and allows for quick and sensitive electrochemical detection.[60,61] Furthermore, the fabricated sensor strip successfully quantitated CQL in urine and serum samples.

Experimental Section

Materials and Methods

The detailed information about the materials, reagents, and apparatus is provided in the Supporting Information.

Design and Fabrication of LSG Electrodes

To prepare the LSG sensor, the electrode patterns for the LSG sensor were first drawn by using computer software, and the PI films were treated with alcohol and distilled water and dried at 60 °C before they were laser-irradiated. A sensor strip was designed with three electrodes (working, reference, and counter). The pre-designed patterns were directly laser-engraved on PI films using a portable laser scribing system (Universal Laser Systems PLS 6.75, laser peak power 75 W) with a wavelength of 10.6 μm, and a pulse duration of ≈14 μs was used to irradiate a PI sheet to produce graphene electrodes as the photothermal process leads to molecular rearrangements in air to form a black carbonized layer, namely LSG.[62,63] Typical power values were between 2.4 and 5.4 W, with pulse frequency (PPI) ranging from 1 to 1000. The PPI feature allows for controlling the number of laser pulses per inch, emitted from the laser gun. Consequently, setting PPI to 100% corresponds to a pulse rate of 1000 per inch. Various experimental settings were tuned, including peak power (15%), speed (10%), PPI (1000 mm), and Z-distance (5 mm). Following the fabrication of the LSGEs on PI sheets, an Ag paste was applied to the reference electrode and cured for 30 min at 60 °C. A PDMS passivation layer was used to precisely define the working electrode (3 mm in diameter), and the layer was cured for 30 min at 85 °C. Afterward, copper tape glued with silver paste was used to connect the LSG electrodes externally for better conductivity.

Functionalization of LSG Electrodes

Scheme depicts the fabrication and functionalization of Nafion/Fe3O4/LSG. A laser scribing process transforms orange-colored PI material into black 3D carbonaceous materials shown in the schematic.[31,56,58] Three-electrode LSG sensors (working, reference, and counter) were fabricated on a single PI substrate with measurements of 2.5 cm (length) × 1.5 cm (width). PDMS passivation was used to separate the working electrode (d = 3 mm) from the rest of the pattern.

Scheme 1

Schematic Illustration of the Preparation of Nafion/Fe3O4 Nanohybrids, Fabrication of LSG Electrodes Using Laser scribing, and Subsequent Modification with Nafion/Fe3O4via Drop Casting to Produce Nafion/Fe3O4/LSG Electrodes

Preparation of Fe3O4 NPs

According to the literature, highly water-dispersible Fe3O4 NPs were synthesized using the co-precipitation method.[64] In the first step, 0.32 M FeCl3 and 0.31 M FeCl2 were mixed vigorously for 30 min in 0.45 M HCl solution. Second, the ferrous/ferric salt mixed solution was added to 250 mL of 0.3 M NaOH solution and vigorously stirred for 3 h at ambient temperature in a N2 atmosphere. After 2 h, the obtained black colloidal Fe3O4 NPs were separated by centrifugation and washed with water. The Fe3O4 NPs were then redispersed in 10 mL of deionized water to obtain Fe3O4 NP stock solution (a total concentration of 0.2 mg/mL) and stored at room temperature for later use.

Fabrication of the Flexible Nafion/Fe3O4/LSG-Modified Electrodes

The LSG working electrode was immersed in a 450 μL solution of 1:1 (v/v) EDC (0.5 M): NHS (0.1 M) for 30 min to activate the carboxylic group on the LGS electrode surface. To make a Nafion/Fe3O4 suspension, 10 μL of Fe3O4 NP solution was ultrasonically mixed with 10 μL of Nafion alcoholic solution (5%) for 45 min. Following this, 8 μL of the Nafion/Fe3O4 suspension was drop-coated on the activated LSG electrode surface, followed by 30 min of incubation to ensure effective adsorption. This ultrasonication treatment produced free radicals in the Nafion polymer chain; later, these free radicals could react with unsaturated C=C groups in the LSG, resulting in a chemically bonded Nafion/Fe3O4/LSG electrode strip.[65] Finally, the electrodes were rinsed in phosphate-buffered saline (PBS, pH 7.0) to remove any residual Nafion molecules, yielding the desired Nafion/Fe3O4/LSG flexible electrode. Later on, the electrodes were stored at 4 °C until they were required again.

Results and Discussion

Surface Morphological Characterization

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the surface morphology of the fabricated LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes. The SEM and TEM micrographs of the various fabricated LSG strip electrodes are shown in Figure a–f, respectively. The SEM image of LSG (Figure a) shows a three-dimensional porous graphene flake structure with a large accessible surface area, which is consistent with previous reports of LSG obtained by laser irradiation of the PI substrate.[59,66] Similarly, the SEM image of the Fe3O4/LSG electrode (Figure b) exhibits that cubical Fe3O4 NPs are well-covered over the LSG flake surface skeleton, particularly at the flake’s edge. Figure c shows the typical SEM image of the Nafion/Fe3O4/LSG electrode, which indicates that aggregated Nafion/Fe3O4 hybrid structures have compactly coated most of the LSG electrode surface, confirming the adsorption of Nafion/Fe3O4 on LSG. A plausible mechanism for an aggregated system within the LSG seems to impregnate Nafion-formed interfacial interactions (hydrogen bonding) between the −COOH group of LSG and −SO3H of Nafion.[67] Also, TEM images of the LSG electrode (Figure d) reveal few-layered graphene flakes with rumpled edges. The rumpled formation is considered to be the result of thermal expansion caused by laser irradiation.[58] Furthermore, the selected area electron diffraction (SAED) pattern for LSG (Figure d inset) reveals distinct poly-crystalline lattice fringes. TEM micrographs of Fe3O4/LSG (Figure e) show that Fe3O4 NPs (∼45 nm average size) are densely anchored over the LSG, and the SAED pattern confirms that the Fe3O4 has poly-crystalline inverse cubic spinel composition (Figure e-insert). Figure f presents a typical TEM micrograph of Nafion/Fe3O4/LSG electrodes, in which Nafion/Fe3O4 interacts incoherently with the LSG matrix, resulting in bigger particles with an average size of 80–120 nm. The inset figure in Figure f displays that the SAED pattern exhibits well-defined diffraction rings, and highly crystalline dots confirm the effective adsorption of the Nafion/Fe3O4 hybrids on the LSG. Besides, the EDX (energy-dispersive X-ray) spectra and elemental mapping reveal that the nitrogen and oxygen elements are embedded uniformly in the LSG electrode (Figure g,j). Similarly, the carbon, oxygen, and iron elements are evenly distributed on the Fe3O4/LSG electrode (Figure h,k). Furthermore, the EDX spectrum and elemental mapping images of Nafion/Fe3O4/LSG (Figure i,l) validate the presence of C, O, Fe, S, and F in its structure. It also implies that the simple ultrasonication method used for the preparation of hybrid nanocomposites is effective.

Figure 1

(a–f) SEM (scale bar for 10 μm) and TEM (scale bar of 200 nm; the inset shows the SEAD pattern) images of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes, (g–l) EDX spectra and elemental mapping images of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes.

In order to determine the accessibility of water-based electrolytes into the electrode, measurements of contact angles (wetting angle) were made on the electrode strip. Thus, we assessed the wettability of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG by measuring the fixed water contact angle. Figure S1 shows the image of a water drop on the surface of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes, and each electrode showed different wetting characteristics. The pristine LSG showed a higher degree of contact angles (90°) that could be attributed to its hydrophobic property. Fe3O4/LSG exhibited a considerably smaller contact angle of 68°, suggesting that micro–nanostructures could be incorporated into 3D network frameworks to increase the hydrophobic properties.[68] More importantly, the Nafion/Fe3O4/LSG strip electrode possesses an excellent wettability behavior with substantially reduced contact angles to 12° due to their polar groups (OH, COOH, and −SO3H), which facilitates electrolyte diffusion.[67]

Structural and Composition Characterization

The X-ray diffraction (XRD) was used to investigate the crystalline structure of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG, and the resulting diffraction patterns are shown in Figure a

Figure 2

(a) XRD patterns for LSG, Fe3O4/LSG and Nafion/Fe3O4/LSG. (b) Raman spectra for LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes.

The XRD pattern of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG sensor strips exhibits a broad, intense diffraction peak centered at 2θ = 25.5° corresponding to the (002) plane, giving an interlayer spacing of 0.38 nm between (002) planes, indicating a high degree of graphitization. Additionally, the broadening depicts a c-axis periodicity of the complex 3D graphitic structure.[31] Furthermore, 2θ = 42.9 corresponds to a peak resulting from reflections (100) associated with in-plane structures. For Fe3O4/LSG and Nafion/Fe3O4/LSG, the major peaks observed at 2θ = 35.48, 37.11, 42.27, 43.12, 53.5, 57.0, 62.62, and 71.05 are assigned to the (311), (222), (400), (311), (422), (511), (440), and (620) facets, respectively, typically corresponding to the simple cubic (sc) structure of Fe3O4 (JCPDS no. 75-0033) along with (002) and (100) planes,[39] which ensures the successful anchoring of Fe3O4 on the surface of LSG.

The Raman spectra of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG are shown in Figure b. As shown in Figure b, there are three prominent peaks observed. The peaks located at 1330 cm–1 (D band) and 1550 cm–1 (G band) correspond to A1g vibrational modes of the sp3-hybridized disordered carbon lattice and the E2g vibration mode in sp2-hybridized ordered carbon lattice, respectively.[69] A 2D peak of the LSG can be observed at 2700 cm–1. The 2D profile is evidence for the formation of a randomly stacked layer of graphene.[70] Additionally, the D band and G band positions and width of the Fe3O4/LSG and Nafion/Fe3O4/LSG are slightly different from LSG as a result of larger structural defects and small graphene domains. ID/IG values of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG are 0.82, 0.89, and 0.96, respectively, which indicate that the −SO3H organic moiety has successfully functionalized LSG.[67] Additionally, the characteristic Raman mode of Fe3O4 can be detected at 616 cm–1, corresponding to the A1g mode of Fe–O vibration, confirming that the composite structure of Nafion/Fe3O4 is anchored on the LSG electrode surface.[58]

We further characterized the functionalization of LSG with Nafion/Fe3O4 using X-ray photoelectron spectroscopy (XPS) spectra. Nafion/Fe3O4 bonded into LSG is evident from signals for C, F, S, Fe, and O in the wide-scan spectra (Figure a). The C1s core-level spectrum (Figure b) shows the characteristic peaks of C=C (284.3 eV), C–O–C (285.2 eV), C–F (288.7 eV), and C–F2 (291.3 eV), which correspond with Nafion bonded on LSG.61Figure c illustrates that the peaks at 161.7 and 163.1 eV belong to the −SO3– side chain within Nafion, and the C–S=O configuration verifies that the sulfonate groups of Nafion are in strong contact with LSG.[71]Figure d shows two peaks at 530.4 and 531.9 eV attributed to the C–O and C=O surface oxygen complex. Fe2p spectra in Figure e demonstrate two intense peaks at 710.2 and 722.3 eV corresponding to the binding energies of Fe2p3/2 and Fe2p1/2 bonds, respectively, resulting in a slight chemical shift difference between Fe2+ and Fe3+ in Fe3O4. In addition, the occurrence of a satellite peak between these two peaks centered at about 712.7 and 717.5 eV can be ascribed to the formation Fe3+ and Fe2+.[72] N2 adsorption/desorption was used to determine the textural properties of the samples. The results are illustrated in Figure S2. It is noted that both LSG and Nafion/Fe3O4/LSG samples have isothermal shapes that fall into both type I and type IV (according to IUPAC classification), indicating a mesoporous and microporous state, respectively.[73]Figure S2 (inset) shows pore size distribution curves, demonstrating that the pores in the synthesized LSG materials are less than 5 nm in size. BET surface areas of 10.65 and 15.36 m2/g with pore volumes of 0.910 and 0.266 cm3 g–1 were observed for LSG and Nafion/Fe3O4/LSG, respectively. Therefore, it can be concluded that after the immobilization of Nafion/Fe3O4 in the LSG matrix, the surface area of the material would increase due to superior physical properties.

Figure 3

(a) XPS survey spectra of Nafion/Fe3O4/LSG electrodes and their (b) C1s, (c) S2p, (d) O1s, and (e) Fe2p core level spectra (inset: high-resolution spectra of Fe2p3/2).

Electrochemical Characterization of the Electrode Strip

To prove the electrochemical characteristics of the fabricated LSG, Fe3O4/LSG, Nafion/LSG, and Nafion/Fe3O4/LSG electrode strips, cyclic voltammetry (CV) performance acquired in 0.1 M KCl containing 1.0 mM Fe(CN)63–/4– as a redox couple at a scan rate of 100 mV/s is shown in Figure a. A pair of well-defined redox peaks with a peak-to-peak separation (ΔEp) of 80 mV (ΔEp = Epa – Epc, where Epa and Epc are the anodic and cathodic peak potentials, respectively) was observed for a pristine LSG electrode, which incorporated a quasi-reversible electron transfer of the Fe3+/Fe2+ redox couple. Despite Fe3O4 NPs being embedded in the LSG surface, the redox peak current intensity was slightly increased Ipa = 18.37 μA (0.203 V) and Ipc = −21.72 μA (0.062 V) than that of pristine LSG. As compared to the LSG electrode, Fe3O4 NP-embedded LSG would provide an enhanced activity surface area and a higher electrocatalytic effect, which would lead to a higher current response. In contrast, when the LSG electrode was modified with a Nafion membrane, the redox peaks were severely suppressed (Ipa = 3.28 μA and Ipc = −6.05 μA) with a peak separation of 141 mV. This is because Nafion can act as a mass transfer blocking layer for the diffusion of [Fe(CN)6]3–/4– into the electrode surface and hinder the electron transfer. After modification with Nafion/Fe3O4/LSG, ion exchange occurred between Fe3O4/LSG and the Nafion membrane, resulting in an increase in the redox peak current [∼twofold higher than that observed with LSG in the potassium ferricyanide solution (Ipa = 27.17 μA and Ipc = −27.73 μA)], indicating that Nafion/Fe3O4/LSG can transfer electrons effectively.[74,75] These results suggest that the Fe3O4 NPs play a critical role in facilitating electron transfer. On one side, the micro–nanostructure confers an accessible active site. Additionally, surface-anchored Nafion/Fe3O4 can enhance electron transfer across electrodes and electrolytes due to their excellent wetting characteristics and superior catalytic activity.

Figure 4

(a) CVs of LSG, Fe3O4/LSG, Nafion/LSG, and Nafion/Fe3O4/LSG electrodes in 1.0 mM Fe(CN)63–/4– and 0.1 M KCl as supporting electrolyte solutions at the 100 mV/s scan rate. (b) CV behavior of Nafion/Fe3O4/LSG in 1.0 mM Fe(CN)63–/4– in 0.1 M KCl at different scan rates (10–100 mV/s). (c) Peak current vs square root of scan rate calibration plots. (d) EIS spectra of LSG, Fe3O4/LSG, Nafion/LSG, and Nafion/Fe3O4/LSG electrodes measured in the frequency range from 100 kHz to 1 Hz in 1.0 mM Fe(CN)63–/4– and 0.1 M KCl electrolyte (inset: Randles-equivalent circuit model).

The electrochemical activity of Nafion/Fe3O4/LSG was investigated further using CV in 0.1 M KCl containing 1.0 mM ferro/ferri redox mediators at various scan rates within the range 10–100 mV/s, as shown in Figure b. The results showed that as the scan rate increased, redox peak currents and peak-to-peak separation potential increased, implying an interfacial behavior with quasi-reversible electron transfer kinetics. Figure c shows that Ipa and Ipc were linearly related to the square root of the scan rate. In accordance with the Randles–Sevick equation (eq ), we calculated the active surface area of surface-functionalized electrodes.where D refers to the typical diffusion coefficient of ferricyanide solution (0.76 × 10–5 cm2 s–1), Ipa, ν, A, C and n refer to the anodic peak current, scan rate (V s–1), active surface area, the concentration of the ferricyanide in bulk solution, and the number of electron transfers (n = 1). Accordingly, the active surface area of LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG is calculated to be 0.129, 0.224, and 0.379 cm2, respectively.

The electron transfer characteristics and interfacial properties of surface-modified LSG electrodes (LSG, Fe3O4/LSG, Nafion/LSG, and Nafion/Fe3O4/LSG) were investigated using the electrochemical impedance spectroscopy (EIS) technique. EIS experiments were carried out in a 1.0 mM ferro/ferricyanide redox system containing 0.1 M KCl at an open-circuit potential ranging from 100 kHz to 1 Hz, and the results are shown in the Nyquist plot (Figure d). In addition, the data were fitted to an equivalent circuit depicted in the inset of Figure d, where W, C, Rs, Qdl, and Rct represent Warburg impedance, capacitance, electrolyte solution resistance, double-layer capacitance, and charge transfer resistance, respectively. The electron transfer resistance is proportional to the semicircular diameter of the impedance curve (Rct).[23] The semicircle domain is found in the bare LSG, indicating a high electron transfer resistance (978.4 Ω). The Rct was reduced to 728.3 Ω after modification with Fe3O4, which was attributed to Fe3O4’s excellent conductivity. By modifying the LSG electrode with Nafion membranes, the semicircle diameter was significantly increased (Rct = 1435.4 Ω). This is due to the fact that the Nafion membrane acts as an electron barrier, introducing resistance into the electrode/electrolyte system, interfering with electron transfer. Besides, the Rct of Nafion/Fe3O4/LSG declined obviously (Rct = 453.3 Ω) when compared to that of LSG, indicating a very good electron-conducting composite. The EIS data were found to be consistent with the CV results (Figure a). These observations revealed that the Fe3O4 NPs can help facilitate electron transport, whereas the negatively charged group (SO3–) present in Nafion provides stronger resistance to anions and bulky molecules, allowing the electrode to more effectively preconcentrate the target cation.

Electrochemical Oxidation of CQL on the Nafion/Fe3O4/LSG Strip Electrode

The electrochemical characteristics of various fabricated strip electrodes toward the oxidation of CQL were evaluated by using CV at a scan rate of 100 mV/s in the absence (dotted line) and presence (solid line) of 400 nM CQL in 0.1 M NaOH (Figure a). Interestingly, in the absence of CQL, all fabricated electrodes did not show any anodic peak current response. In contrast, in the presence of CQL, a sharp and well-defined anodic peak current for CQL oxidation was obtained for all electrodes. There is a significant oxidation peak observed in the CV of bare LSG, corresponding to a peak current of 11.31 μA at 0.42 V. The LSG modified with Fe3O4 exhibits improved electrocatalytic performance, with the lowest peak potential at 0.41 V and a higher anodic current intensity of 13.5 A. The outstanding catalytic activity and high electrical conductivity of Fe3O4 are responsible for this significant increase in current. In addition, the Nafion/Fe3O4/LSG CV curve displays a clearly pronounced and enhanced oxidation response with a peak intensity of 20.14 μA at 0.42 V. It is noteworthy that the anodic peak current of CQL at Nafion/Fe3O4/LSG is two times greater than that at bare LSG. The Nafion and Fe3O4 composite combined showed a clear synergistic effect that transduced electron transfer and intensified specific surface areas at the electrode interface, thus enhancing the current response signal.

Figure 5

(a) Cyclic voltammograms obtained for absence (dotted line) and presence (solid line) of 400 nM CQL in 0.1 M NaOH at LSG, Fe3O4/LSG, and Nafion/Fe3O4/LSG electrodes. (b) CV responses of various scan rates (10–100 mV/s) at Nafion/Fe3O4/LSG electrodes with the presence of 400 nM CQL in 0.1 M NaOH. (c) Linear plot of oxidation peak current vs scan rates. (d) Relationship of peak potential vs scan rates. (e) Proposed electro-oxidation mechanism for CQL on Nafion/Fe3O4/LSG electrodes.

Influence of the Scan Rate

The effect of the scan rate (10–100 mV/s) was investigated on the electrochemical oxidation of CQL (400 nM) at the Nafion/Fe3O4/LSG-modified electrode (Figure b). In the examined ranges, the oxidation peak current for CQL was directly related to the scan rate. Furthermore, it is observed that a good linear relationship is observed when plotting the current against the scan rate (Figure c). Ipa (μA) = 0.163 [mV/s] + 5.65 was found to be the linear regression equation, with a correlation coefficient of R2 = 0.993, suggesting a diffusion-controlled electrode reaction of CQL at the Nafion/Fe3O4/LSG electrodes.[76,77]Epa and υ relationship can be defined using Laviron’s equation (eq ) in the context of a diffusion-controlled electrochemical irreversible process.[60]

Based on the Epa versus υ relationship shown in Figure d, the value of E0′ is anticipated to be 0.407 V by prolonging the curve to υ = 0. The electron transfer coefficient (α) and electron transfer rate constant (K0) were calculated from the slope of the Epa versus ln υ plot to be 0.40 and 0.618 cm/s, respectively. This K0 value is twofold higher than that of pristine LSG (0.232 cm/s), indicating rapid electron transfer at the developed interface.

The number of electrons (n) consumed in the electro-chemical oxidation of CQL on Nafion/Fe3O4/LSG was calculated from the equation (eq ) for irreversible reactions[78]where α is the charge transfer coefficient, n is the number of electrons consumed in the oxidation process, and Ep/2 is potential at the half-peak current. Thus, the number of electrons consumed in the process could be calculated to be 0.94 (∼1 electron). A voltammetric electrochemical mechanism for the electro-oxidation of CQL at Nafion/Fe3O4/LSG in an alkaline solution is represented in Figure e. Initially, the CQL was oxidized by one electron and one proton, resulting in phenoxy radicals, followed by an addition reaction with OH that generated unstable semiquinone anion radicals. As a result of the fast nature of the addition reaction, it was not possible to detect the cathodic voltammetry peak associated with the reduction of the phenoxy radical. Finally, the semiquinone intermediate underwent a disproportionation reaction to generate CQL and a p-quinone intermediate.[3,4,11,79]

Influence of Concentration

Figure S3a shows the SWV (square-wave voltammetry) response of CQL (10–100 nM) on Nafion/Fe3O4/LSG with consecutive injection of 10 nM CQL to 0.1 M NaOH at a scan rate of 20 mV/s. The amplitude of the anodic current response peaks with an increase in CQL from 10 to 100 nM would suggest that the sensor could recognize the CQL without fouling. Anodic peak currents and CQL concentrations are linearly related, as shown in Figure S3b. The good correlation coefficient (R2) 0.996 and the linear regression equation Ipa (μA) = 0.276 [CQL (nM)] + 8.626 imply that CQL oxidation is controlled, which is consistent with an earlier report.[2−4] Furthermore, the plot of the logarithmic peak current against the concentration of CQL (Figure S3c) is a straight line and corresponds to a regression equation of Ipa (μA) = 0.455 [CQL (nM)] + 0.536 (R2 = 0.992). Based on these findings, the catalytic reaction in the Nafion/Fe3O4/LSG sensor is driven by first-order kinetics.

Effect of Modifier Amount

The volume of nanomaterials immobilized on the LSG electrode is crucial because it affects the electrode’s sensitivity significantly. To investigate the impact of loading-level optimization, different amounts of Nafion/Fe3O4 ranging from 2 to 12 μL were used to modify LSG to fabricate the sensor, and their sensing ability was tested toward 400 nM CQL drug detection (Figure S3d). It is clear that increasing the amount of catalyst loading up to 8 μL increased the oxidation peak current density due to an increase in recognition sites on the electrode surface. Furthermore, increasing the catalyst loading on the electrode above the optimum value reduces the peak current density response. These findings indicated that using more than 8 μL results in a thicker modifier layer, which has a negative impact on interfacial electrode transport between solution and electrode surface. Therefore, 8 μL of drop-coated Nafion/Fe3O4/LSG was used for further electrochemical experiments.

Effect of Applied Potential

Working potential is yet another important factor that influences the electrochemical response of sensors. To optimize the working potential, the influence of applied potential was initially explored from the amperometric response of CQL on the Nafion/Fe3O4/LSG electrode in the range of 0.15–0.30 V, and the corresponding amperometric responses upon successive injection of 0.01 μM CQL in 0.1 M NaOH solution under stirred circumstances are recorded in Figure S3e. As can be seen, as the applied potential increases from 0.15 to 0.30 V, the anodic current step increases. When compared to 0.25 V, the current response at 0.30 V decreases significantly. An ideal applied potential for amperometric detection of CQL was chosen based on the most intense current response for the oxidation of CQL at 0.25 V for subsequent studies.

Quantitative Assay of CQL

The quantitative assay of CQL was investigated by recording the differential pulse voltammetry (DPV) and chronoamperometry (CA) response of various concentrations of CQL at Nafion/Fe3O4/LSG. Figure a shows the DPV performance of Nafion/Fe3O4/LSG as a function of various concentrations of CQL (from 0.001 to 100 μM) in a potential window of 0–0.6 V. The oxidation currents rose directly in proportion to the CQL concentration. Calibration curves for CQL concentrations versus oxidation peak currents are linear (Figure b). As illustrated in Figure b, two linear ranges (0.001–10 μM and 10–100 μM) for the oxidation of CQL can be observed at the Nafion/Fe3O4/LSG. The corresponding linear equations are I (μA) = 0.929 [CQL (μM)] + 17.4 (R2 = 0.990) and I (μA) = 0.050 [CQL (μM)] + 13.1 (R2 = 0.991). Based on the standard deviation of the control sample (RSD) and the slope of the fitted calibration plots [LOD = 3 (RSD/S)], we estimated the limit of detection (LOD) to be 0.73 nM.

Figure 6

(a) DPV signals obtained for successive additions of CQL (0.001–100 μM) in 0.1 M NaOH at 50 mV/s on Nafion/Fe3O4/LSG. Inset: magnified view of the lower CQL concentration vs oxidation peak currents. (b) Linear calibration plots of oxidation peak currents vs the CQL concentrations. Inset: Linearity plot for lower CQL concentration vs peak current. The error bars represent ± SD and n = 3 samples, with a maximum RSD of 2.41%. (c) CA responses of the Nafion/Fe3O4/LSG sensor in 0.1 M NaOH with successive additions of CQL at 0.25 V applied potential. Inset: the amplified i–t curve for CQL at low concentrations. (d) Calibration curves for current vs concentration of CQL. Inset: expanded ranges of selected concentrations. The error bars represent ±SD and n = 3 samples, with a maximum RSD of 2.68%.

In addition, the amperometric (i–t) method was adopted for the quantitative analysis of CQL because it possesses good selectivity, excellent sensitivity, wide linear range, trace level threshold, and minimal electrode passivation. Figure c shows the amperometric response (i–t curve) of Nafion/Fe3O4/LSG to successive injections of CQL into 0.1 M NaOH solution at an optimized applied potential (0.25 V). Each addition of CQL results in well-defined and steady amperometric responses. As shown in Figure c, response currents progressively increase as CQL concentration increases from 0.001 to 10 μM. As CQL concentrations increase from 0.001 to 10 μm at 0.25 V, it is revealed that the sensor produces a larger response current, and significant background noise causes an aggrandized step current. This behavior is consistent with earlier reports.[3,36] Similarly, two dynamic linear ranges of CQL from 0.001 to 1.0 and 1.0 to 10 μM (Figure d), the linear regression equation could be expressed as Ip (μA) = 0.212 [CQL (μM)] + 6.60 (R2 = 0.998) and Ip (μA) = −0.176 [CQL (μM)] + 6.96 (R2 = 0.993), respectively. Furthermore, based on the abovementioned formula, the LOD corresponding to three times the average blank value’s RSD (relative standard deviation) was calculated to be 0.73 nM, and sensitivity was predicted to be 17.4 μA μM–1 cm–2. The electro-catalytic performance of our sensor was superior or comparable to that of other modified electrodes reported previously, as shown in Table . Based on the comparison table, the fabricated sensor offers commendable performance for detecting CQL in simple processing, high stability, and low detection limits. Moreover, these different sensor platforms for CQL quantification typically used macroelectrodes [glassy carbon electrode (GCE)[11,12,36] and screen-printed carbon electrode (SPCE)][3,4] that were decorated with graphene/metal or metal oxide NPs to improve sensitivity. Most of the preparation processes for these similar nanostructures are complicated due to their multistep fabrication process with less controlled repeatability, and the high preparation costs involved. Although they cannot be used for the POC testing due to the larger size of the electrochemical system consisting of external reference and counter electrodes.

Table 1

Analytical Comparison of the Proposed Method and Some of the Previously Reported Electrochemical CQL Biosensors

modified electrode	technique	linear range (μM)	LOD (nM)	references
aBSA-AuNC-Cu2+	fluorescence	1–12	630	(1)
Sn(MoO4)2/bGCE	DPV	0.05–234	14	(2)
MCO/S-rGO/cSPCE	i–t	0.001–84	0.9	(3)
CeVO4/SPCE	DPV	0.02–215	4.0	(4)
dSQD	fluorescence	0.024–30	15	(10)
GO@LaVO4–NCs/GCE	DPV	0.025–438.25	2.44	(11)
N-eCQD@Gd2O3/GCE	i–t	0.3–220	2.1	(36)
Nafion/Fe3O4/fLSG	DPV, i–t	0.001–100	0.73	this work

Bovine serum albumin–gold nanoclusters quenched by Cu2+.

Glassy carbon electrode.

Screen-printed carbon electrode.

Sulfur quantum dots.

Carbon quantum dot.

Laser-scribed carbon electrode.

Specificity Studies

In the analysis of real samples, selectivity plays a key role. Therefore, we investigated the selectivity of our fabricated sensor by measuring its response (100 nM) via DPV by introducing several interferences, as shown in Figure a and b. The results illustrated that the presence of 100-fold excess of various common metal ions (Zn2+, Mg2+, Cu2+, Pb2+, Cd2+, K+, and Na+) and some biologically active molecules (dopamine, uric acid, ascorbic acid, glucose, uric acid, glycine, hydroquinone, and caffeine) in 50-fold excess, were not significantly affected. Furthermore, 20-fold excess concentration of some pharmaceutical drugs (flutamide, metronidazole, acetaminophen, and amoxicillin) also exhibited negligible (<5%) interference with the CQL test. Therefore, Nafin/Fe3O4/LSG electrodes can be used in electrochemical sensors or pharmaceutical formulations to detect CQL in a selective way.

Figure 7

(a) DPV response of CQL oxidation in the presence of interfering species with 100 times excess of common metal ions (Zn2+, Mg2+, Cu2+, Pb2+, Cd2+, K+, and Na+); a 50-fold excess of physiological interference (dopamine, uric acid, ascorbic acid, glucose, uric acid, glycine, hydroquinone, and caffeine); and a 20-fold excess of widely prescribed drugs (flutamide, metronidazole, acetaminophen, and amoxicillin). (b) Percentage of interfering-species current signals compared to CQL. (c) CV response of the Nafion/Fe3O4/LSG sensor in 0.1 M NaOH containing 400 nM CQL at different time intervals. (d) Steady-state response observed at Nafion/Fe3O4/LSG for 100 nM CQL in 0.1 M NaOH up to 3000 s. The applied potential is +0.25 V. (e) Reproducibility of Nafion/Fe3O4/LSG for six separate replicates and (f) repeatability of Nafion/Fe3O4/LSG for nine repeated measurements at a single electrode in 400 nM CQL in 0.1 M NaOH at 100 mV/s. (g) Typical CV curves for Nafion/Fe3O4/LSG for 400 nM CQL at 50 mV/s in 0.1 M NaOH as supporting electrolyte in flat, convex, and concave bending modes. Inset: Schematic representation of Nafion/Fe3O4/LSG in flat, convex, and concave bending stress modes, respectively. (h,i) SWVs recorded at Nafion/Fe3O4/LSG sensors after sequential addition of CQL-spiked human blood serum (10–100 nM) and human urine samples (5–100 nM) into 0.1 M NaOH at 50 mV/s.

Stability, Reliability, and Repeatability Measurements

In order to make the developed sensor feasible and applicable, the stability and reproducibility are of paramount importance. The stability of the Nafion/Fe3O4/LSG sensor was assessed by storing the sensor for 15 days under ambient conditions and then performing voltammetry measurements in 400 nM CQL solution (Figure c). The RSD of the sensor was 3.46%, indicating that it is very stable over time. In addition, another method for examining the stability of the sensor response toward CQL oxidation was evaluated by amperometric technique with CQL at 100 nM for 3000 s (Figure d), implying that the strip electrode exhibited a long-term electrochemical stability and had no surface fouling. Reliability of the Nafion/Fe3O4/LSG sensor was assessed through repeated voltammetric measurements on six replicate sensors (Figure e). They had good reproducibility with an RSD of 2.37% for 400 nM CQL solution. Furthermore, a series of nine CV measurements using the same Nafion/Fe3O4/LSG electrode in 0.1 M NaOH containing 400 nM CQL (Figure f) revealed an RSD value of 2.51%, indicating that the modified electrode has good reproducibility.

Versatility Test

To determine the impact of bending stress on electrochemical reactions in sensors, CV testing was used to examine the rigidity of Nafion/Fe3O4/LSG electrodes. Hence, the electrochemical properties of the flexible device were evaluated under flat, convex, and concave bending stresses to simulate various deformations that may occur in practice. CV measurements are performed on each sensor in 0.1 M NaOH contains 400 nM CQL at 50 mV/s, as shown in Figure g. It was observed that the electrochemical responses of Nafion/Fe3O4/LSG electrodes were not affected by convex and concave bending. Furthermore, the electrode responses remain almost the same, showing that the new electrode platform is flexible.

Detection of CQL in Spiked Human Urine and Blood Serum Samples

A practical application of the Nafion/Fe3O4/LSG strip electrode has been explored by examining the standard addition method to determine CQL in human blood serum and urine samples. To accomplish this, human blood serum was purchased from Sigma-Aldrich, Taiwan, and fresh urine samples were obtained from healthy individuals at National Taipei University of Technology, Taiwan. Human urine samples were centrifuged for 10 min at 5000 rpm, filtered, and the supernatant urine was obtained. On other hand, the serum sample was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Both serum and urine supernatant samples (250 μL) were diluted in 25 mL of 0.1 M NaOH and stored in a refrigerator for further analysis. The dilution can actually help reduce the matrix effect of real samples. A known quantity of blood serum and human urine diluted samples was spiked separately with CQL at different concentrations using the standard addition method, and SWV curves are recorded (Figure h,i). The analytical results are presented in Table S1. The estimated recovery of CQL in the spiked urine and blood serum samples were found to be in the range of 97.0–102 and 98.0–102.6%, respectively, with an RSD less than 2.88% (n = 3), indicating that the electrode is feasible and reliable for detecting CQL in blood serum and urine samples. Figure S4 shows the SWV response for the determination of CQL in human urine samples spiked at 100 nM over commercial AuEs (gold electrodes) and a Nafion/Fe3O4-modified AuE to validate the accuracy of the newly proposed sensor. The results showed that the Nafion/Fe3O4/LSG electrode had a superior electrochemical performance over a commercial Au electrode and a modified Nafion/Fe3O4/AuE electrode, indicating that the new procedure is reliable and feasible.

Application of Portable CQL sensors

We demonstrate the use of Nafion/Fe3O4/LSG sensors for analysis of small sample volumes and POC testing for the detection of CQL by the standard addition method using a portable electrochemical workstation (Figure S5a). Furthermore, to determine the relationship between peak current responses and CQL concentrations on a portable mini-workstation, SWV was applied (Figure S5b). Results from the portable mini-station are comparable to those from traditional large-scale workstations. A linear relationship exists between peak currents and CQL concentrations. From Table S2, we can see that satisfactory recoveries range from 91.2 to 99.35%, which indicates that the proposed flexible sensor for intelligent detection of CQL in real samples is satisfactory and applicable.

Conclusions

In this work, we report the development of a new flexible strip electrode by immobilizing Nafion/Fe3O4 nanohybrids on a porous LSG electrode. LSG was fabricated on commercial PI using a direct laser scribing process. A highly ambient stable Nafion/Fe3O4 nanohybrid was synthesized using an ultrasonic-assisted technique. The proposed flexible electrode was fabricated by drop coating Nafion/Fe3O4, which is electrostatically self-assembled between positively charged Fe3O4[80,81] and negatively charged Nafion on LSG flexible substrates. In addition, the electrodes were characterized by various spectroscopy and analytical methods. Owing to their porous 3D structure, an enriched surface area at the active edges and an excellent wettability in water contributed to the impressive kinetics of electron transfer reactions. Specifically, the 3D hierarchical structure of flexible LSG provides a larger specific surface area and higher mechanical strength than traditional electrodes. This electrode was used to construct a flexible transducer for ultrasensitive clioquinol detection, and it performed admirably in terms of a wide linear range (1 nM to 100 μM), low detection limit (0.73 nM), and high stability. In addition, it was demonstrated that this analytical method proved effective even at interferences that were 50-fold greater than the measured value. The detection of clioquinol in human serum and urine samples using the fabricated sensor shows that it can be used as an effective clinical diagnosis in in vivo studies.