1. Introduction
The development of high-sensitivity biosensors relies heavily on the optimization of carbon nanotubes (CNTs), specifically focusing on enhancing their dispersity and electrochemical properties while maintaining proper electrical conductivity [
1,
2]. Achieving well-dispersed CNTs is essential to prevent agglomeration, ensuring uniform coverage and accessibility of the nanotube surface for biomolecule attachment [
3]. Biosensors developed with well dispersed and optimized CNTs have the potential to detect biomolecules with enhanced sensitivity and rapidity. Diabetes mellitus is a chronic metabolic disorder affecting millions of people worldwide [
4,
5,
6]. Non-invasive glucose monitoring has emerged as a highly sought-after alternative to blood-based measurements, offering continuous monitoring without the need for repeated finger pricks [
7]. The development of reliable biosensors capable of non-invasive detecting glucose in sweat is of significant interest for diabetes management [
8].
Carbon nanotubes (CNTs) have attracted significant attention due to their exceptional physical and chemical properties that has prompted increasing number of researchers for the construction of an analytical device to detect biomolecules ranging from glucose, nucleic acids, and small proteins, and prokaryotic and eukaryotic bacterial cells [
9]. They have a tubular structure composed of monomer graphene deposed either in a single sheet (single-walled carbon nanotube, SWCNT) or in multiple sheets of graphene (multi-walled carbon nanotube, MWCNT) [
10].
Single-walled carbon nanotubes (SWCNTs) have emerged as a versatile nanomaterial for biosensing applications, owing to their exceptional electrical, mechanical, and chemical properties [
11]. SWCNTs possess high electrical conductivity, a large surface area, and excellent biocompatibility, making them ideal candidates for glucose sensing [
11,
12]. These nanotubes have a diameter typically in the order of nanometers, with lengths ranging from micrometers to millimeters that lead to a wide range of potential new research applications [
13]. The discovery of SWCNTs, along with their multi-walled counterparts (MWCNTs), has opened new avenues for innovation and applications in nanotechnology [
12]. Multi-walled carbon nanotubes (MWCNTs) have garnered significant attention in the field of biosensor research due to their unique properties including electrical conductivity, increased surface area, and biocompatibility [
14]. Unlike SWCNTs, MWCNTs consist of several nanotubes nested within one another, with the number of layers typically ranging from two to a few dozen [
15]. This unique structure imparts distinctive properties to MWCNTs, making them valuable across a wide range of applications in various scientific and industrial fields.
By leveraging the specific interaction between glucose and enzymes immobilized on SWCNT surfaces, the detection of D-glucose can be achieved with remarkable sensitivity and selectivity [
12]. The proposed SWCNT-based biosensor offers several advantages for non-invasive glucose monitoring in sweat [
13]. Firstly, the high electrical conductivity of SWCNTs enables efficient charge transfer, facilitating sensitive and rapid glucose detection. Secondly, the large surface area of SWCNTs provides ample sites for enzyme immobilization, enhancing the biosensor's sensitivity and detection limits [
8]. Moreover, the biocompatibility of SWCNTs ensures long-term stability and compatibility with sweat constituents, enabling reliable and accurate glucose measurements [
14].
Furthermore, the unique mechanical properties of SWCNTs make them suitable for the development of miniaturized biosensor devices, which are highly desirable for point-of-care diagnostics [
12]. In this research paper, we highlighted the development and characterization of a SWCNT-based biosensor for D-glucose detection in sweat. The fabrication process involves the functionalization of SWCNTs, followed by the immobilization of glucose oxidase (GOx) as the sensing element [
15]. The performance of the biosensor is systematically evaluated using standard glucose solutions, as well as sweat samples collected from healthy individuals. The objectives of this study are to determine the dispersity of CNTs into the solvents for biosensor or sensor development; optimize various parameters, including enzyme loading, pH, and temperature, and enhance the biosensor's performance; and assess or evaluate response of the biosensor to potential glucose interferents with rapidity and sensitivity.
In addition to the remarkable properties of carbon nanotubes, the use of chitosan, a natural polymer, has been gaining traction in the field of biosensing due to its excellent film-forming ability and biocompatibility [
16]. Chitosan offers a favorable microenvironment for enzyme immobilization, significantly affecting the stability and activity of the immobilized enzymes such as glucose oxidase (GOx) [
17]. The integration of chitosan with carbon nanotubes has been shown to enhance the dispersion of CNTs further and prevent their agglomeration, thereby maintaining the high surface-to-volume ratio crucial for enzyme attachment and biosensor sensitivity [
18].
Furthermore, chitosan's cationic nature under acidic conditions facilitates a strong electrostatic interaction with negatively charged biomolecules and enzymes. This interaction not only helps in the immobilization of enzymes on the biosensor's surface but also stabilizes the immobilized enzymes, maintaining their bioactivity over a longer period [
19]. This innovative approach of combining chitosan with CNTs for enzyme immobilization can lead to the development of biosensors with enhanced performance, higher sensitivity, and greater stability.
Moreover, chitosan is known for its non-toxicity, biodegradability, and biocompatibility, making it an excellent material for applications in biosensors, particularly those intended for medical and food industry applications. The presence of chitosan in the matrix of carbon nanotubes-based biosensors can significantly improve the adherence and uniformity of enzyme layers, resulting in more reproducible and reliable glucose detection [
20].
2. Materials and Methods
2.1. Chemicals
SWCNT and MWCNT (called CNTs) were supplied by (Sigma Aldrich, Seoul Korea) and their purity was greater than 95%. The solvents, namely N, N-Dimethylformamide (DMF), Acetone, Di-water, and ethanol were obtained from (Sigma Aldrich, Seoul Korea). The glucose substrate (ng/mL, naturally purified and prepared in) and glucose oxidase enzyme against glucose were procured from Inc. (Sigma Aldrich, Seoul Korea). A 10% (v/v) phosphate buffer saline (PBS, 0.1 M, pH 7.4, 0.8% NaCl) was purchased from Life Technologies (Seoul Korea) and was prepared by mixing with purified water. Glucose substrate was diluted for use in 10% PBS. All other chemicals were of analytical reagent grade and were used without further purification.
2.2. Solvent suspension and Dispersion of CNTs
In this study, the commonly used solvents for preparing spinning solutions included dimethylformamide (DMF), acetone, ethanol, and deionized water (Di-water). The dispersion of the CNTs (SWCNTs or MWCNTs) were performed by mixing CNTs with DMF, acetone, ethanol, and DI-water, individually, at the concentration of 0.5 g/L and sonicated for 2 h for preparing CNTs’ spinning solutions using ultrasonic waves (write the equipment details). After that, the physical properties of dispersed CNTs’ solution like concentration measurement using high UV-spectrophotometer were performed to confirm their effective chemical interaction and high reproducibility with the applied reactant spinning solvents.
The dispersion of CNTs were primarily assessed through sedimentation process by following the previous procedure with minor modifications [
16]. The sediment CNTs in the sonicated solution were captured and monitored with hand-held smart photometer after one day, one week and a month to observe the long-term stability of the dispersions. UV-spectroscopy analysis was performed using a SHIMADZU instrument at 100.0 kV. Additionally, to further understand the interactions between the nanotubes and solvents, UV measurements were conducted. These measurements were used to determine the surface charge of the nanotubes in different solvents, which is a critical factor in understanding their dispersion and stability [
16]. The comprehensive analysis offered valuable data optimizing solvent choice and processing conditions for MWCNT applications in various fields, including sensor technology, nanocomposites, and electronic devices.
2.3. Biosensor Development with SWCNTs
Biosensor was developed with SWCNTs by following procedure with slight modification [
17]. Firstly, different concentrations of SWCNTs (0.025, 0.05, 0.1, 0.2, 0.4 g/L) were dispersed in DMF solution and sonicated for at least 2 h. Following sonication, each sonicated SWCNTs with an aliquot of 10 µl was applied to the sensor plate and annealed for 15 min into incubator at 80 ᵒC to bind the SWCNTs on the sensor plate. After annealed, the biosensor plates were washed with deionized water to eliminate unbound SWCNTs and dried with N
2 gas. Following this, electrical resistance of the biosensor with respect to each concentration was measured using a multi-meter tester to evaluate the impact of SWCNT concentrations on conductivity.
To optimize PBSE, the developed SWCNT-based biosensor was functionalized with different concentrations of a PBSE linker (1.0, 2.0, 4.0, 6.0, 8.0 g/L) to investigate how the linker concentration affects the overall conductivity and stability of the dispersion. Each concentration was thoroughly tested to determine the most effective ratio of linkers for enhancing the electrical properties of the biosensor. In addition, to optimize glucose oxidase (Gox) as a binding substrate, the linker functionalized biosensor was carefully immobilized with Gox at varying concentrations (0.01, 0.05, 0.1, 0.2, 0.5 g/L) and studied its effect on the electrical properties of the composite material. The immobilization of Gox at different concentrations was crucial for determining the optimal amount required to achieve the best sensor performance. To further enhance enzyme immobilization, a solution mixing method was employed, where chitosan was dissolved with the enzyme solution before applying to the SWCNT-based biosensor, ensuring a uniform and stable attachment of glucose oxidase onto the biosensor surface [
21]. The immobilization of Gox at different concentrations was crucial for determining the optimal amount required to achieve the best sensor performance.
2.4. Detection of Glucose by the Fabricated Biosensor
The Gox immobilized biosensor was tested with 80 μL of diluted glucose solution over the range of 0.2 – 0.9 g/L at room temperature using a detection time of 30 min to allow for the Gox-glucose reaction to occur. After the reaction, the applied biosensor was washed with DI water to remove any unbound glucose molecule from the sensor surface, and the resistance of the sensor electrode was measured using a potentiostat. A change in resistance confirmed the attachment of glucose onto the Gox and this change in resistance was only observed for glucose concentration. In this study, linear sweep voltammetry (LSV) measurements were performed at each step using a potentiostat device (DY2013, EG Technology, Seoul, South Korea) at room temperature to measure resistance.
4. Conclusions
This study has investigated the dispersion characteristics of CNTs in various solvents, their interaction with PBSE linkers, the optimization of glucose oxidase (Gox) concentration, and the consequent impact on electrical resistance in biosensor applications. The optimal dispersion of CNTs was achieved in DMF, which exhibited the most stable and uniform nanotube suspension, as visually confirmed and quantitatively supported by UV-Vis spectral analysis. The electrical resistance measurements further validated that DMF is the solvent of choice for SWCNT-based biosensors due to its favorable interaction with the carbon nanotubes. The interaction with PBSE linkers and Gox concentration offered a concentration-dependent electrical resistance, which peaked and then plateaued, indicating optimal ranges for biosensor performance. For glucose detection, the SWCNT-based biosensor exhibited enhanced sensitivity at lower concentrations, suggesting its efficacy for blood glucose monitoring within the physiological range. This research underscores the importance of fine-tuning SWCNT, PBSE, and Gox concentrations to achieve biosensors with optimal electrical properties for medical diagnostics. This study features the SWCNT-based biosensor's potential for accurate and reliable blood glucose monitoring, which is essential for diabetes management. The controlling concentration of SWCNTs, PBSE linkers, and Gox is critical for optimizing the electrical properties of SWCNT-based biosensors. These insights pave the way for the design of highly sensitive and specific biosensors, which are of paramount importance in the fields of medical diagnostics and bioelectronics.