Enhanced Electrocatalytic Properties of Co₃O₄ Nanocrystals Derived from Hydrolyzed Polyethyleneimines in Water/Ethanol Solvents for Electrochemical Detection of Cholesterol

Abstract

The present study describes the effect of hydrolysis of polyethyleneimines in water/ethanol mixture on the morphology of the cobalt oxide (Co₃O₄), used as the main sensor component. The structure of the generated Co₃O₄ nanocrystals is consistent with a well-defined cubic phase crystallography, having only cobalt and oxygen elements. Developing simple, low-cost, sensitive, and selective cholesterol biosensors is essential for accurate monitoring of cholesterol to avoid cardiovascular diseases. These nanocrystals exhibit large surfaces suitable for facile and high loading of cholesterol oxidase enzyme through the physical adsorption method. Then, the fabricated cholesterol oxidase/ Co₃O₄ nanocrystals composite was implemented for potentiometric detection of cholesterol in 10 mM phosphate buffer of pH 7.3. Importantly, the presented cholesterol biosensor revealed a wide linear range of 0.005 mM to 3.0 mM with a limit of detection (LOD) of 0.001 mM. Additionally, the sensitivity of biosensor was estimated around 60 mVdec⁻¹. The selectivity, stability, reproducibility, and repeatability were also observed as satisfactory. The dynamic response of the proposed method demonstrated a fast response time of less than 1 s. Furthermore, the successive addition method confirmed a remarkably stable response towards various cholesterol concentrations. Thus, the developed cholesterol oxidase/ Co₃O₄ nanocomposite may be used as an efficient alternative method to monitor low cholesterol concentrations form real samples.

1. Introduction

Cholesterol represents an essential class of sterol that is found in the human body, particularly in the liver [1]. The function of cholesterol is to regulate bile acids, vitamin D, and glucocorticoids including estrogen and progesterone. The presence of 70% of cholesterol in the human blood is an ester and approximately 30% is free cholesterol content in the human body, thus confirming the total concentration of cholesterol [2]. The physiological level of cholesterol in the human blood should be lower than 5.2 mM, and the cholesterol concentration of 6.2 mM indicates the symptom of hypercholesterolemia. These high levels of cholesterol are causing cardiovascular diseases, myocardial infarction, and atherosclerosis. However, a low level of cholesterol is known as hypercholesterolemia and becomes a cause of anemia and hepatopathy [3,4]. Therefore, it is very essential to monitor cholesterol level to avoid health complications in the human body. Cholesterol has been determined by different analytical techniques including high-performance liquid chromatography (HPLC) [5], colorimetry [6], spectrophotometry [7], and electrochemical routes [8]. The electrochemical techniques are more favorable for the cholesterol analysis due to their fast response detection of cholesterol in low volumes with acceptable stability and reproducibility. Several electrochemical biosensors have been designed for the detection of cholesterol using an enzymatic approach [9]. The use of cholesterol enzymes enables the highly sensitive and wide linear range detection of cholesterol concentration.

Cholesterol + O₂ + ChOx → cholestene-3-one + H₂O₂

(1)

The oxidation of cholesterol in the presence of oxygen and cholesterol oxidase results in cholestene-3-one and H₂O₂. The quantified amount of H₂O₂ gives indirect information about the concentration of cholesterol. Irrespective of high sensitivity of amperometric methods, they have a limitation of interference caused by vitamin C and uric acid at high anodic potential during cholesterol determination [10]. Beside this, the potentiometric method does not use any high potential and it completely prevents the interference effect from ascorbic acid and uric acid during the sensing of cholesterol. The potentiometric method requires a highly active surface for the immobilization of cholesterol oxidase and, consequently, a strong output potential should be produced. Further, the potentiometric method is capable of working within the human body cell for monitoring cholesterol levels; therefore, this electrochemical method is more suitable for the real biological environment [11]. Significant progress in the development of cholesterol biosensors has been made recently. However, there is still ample space to design and fabricate fast and efficient electrochemical cholesterol biosensors via introducing favorable nanostructured materials, which can act as excellent host surfaces for the immobilization of cholesterol oxidase. The low dimension materials have proved to be very fast in response time, high selectivity, and sensitivity towards the development of functional sensing devices [12]. The functional properties of nanostructured materials are mainly governed by the morphology, composition, and size [13,14,15,16,17,18,19]. Among the various nanostructured materials, cobalt oxide (Co₃O₄) is widely studied due to its local and tunable electronic properties. Moreover, the spinel structure of Co₃O₄ allows it to act as a notable material for the fabrication of novel electrochemical devices [20]. Therefore, Co₃O₄ has been utilized in different applications such as heterogeneous catalysis and electrochemical devices [21,22,23,24,25,26,27]. Numerous morphologies of Co₃O₄ have been prepared including nanowires/nanorods [28], nanotubes [29], nanocubes [30], nanospheres [31], and nanoplates [32]. These nanostructures were prepared by various growth methods. Importantly, Co₃O₄ exhibits a high isoelectric point value of approximately eight and it can be increased at high temperatures [33]. The high isoelectric point value allows Co₃O₄ to be an excellent host material for the immobilization of enzymes/antibodies via strong electrostatic forces. The low isoelectric point value of cholesterol oxidase of 4.6 reveals a strong bonding with the nanostructured surface of Co₃O₄ [18]. The oxidation reaction of cholesterol substrate is slow and it should accelerate when an enzyme-based approach is used.

The proper immobilization of cholesterol oxidase onto desired metal-oxide surfaces could lead to a massive increase in the sensitivity, swiftness, and selectivity towards cholesterol detection. For this purpose, we have produced nanostructures of Co₃O₄ using a mixed solution of polyethyleneimines in ethanol/water mixture. There were two aspects of using this approach for the growth of Co₃O₄ nanostructures: first, the polyethyleneimine provided the additional amount of amine groups required for binding the cobalt ions during the growth process in addition to the hydroxyl groups. Second, it can control the growth kinetics of Co₃O₄ due to the use of an organic/aqueous bi-solvent system, thereby resulting in a well-defined morphology. These two aspects together produced a sensitive and selective Co₃O₄ nanocrystals-based cholesterol biosensor. There is no report on the use of the bi-solvent approach to produce Co₃O₄ nanocrystals with enhanced electrocatalytic properties.

In this study, we have prepared nanocrystals of Co₃O₄ using polyethyleneimine as an additional hydrolyzing agent during wet chemical methods. These Co₃O₄ nanocrystals were employed as a host material for the loading of cholesterol oxidase via electrostatic attraction, and the resultant cholesterol oxidase/Co₃O₄ nanocomposite was successfully used for the development of a potentiometric biosensor. The linear range of the proposed method was found between 0.005 mM and 3 mM with a Nernstian slope of 60 mV per decade. The limit of detection (LOD) was estimated to be about 0.001 mM. The selectivity and stability were also studied. The method is highly reproducible, repeatable, and fast in response time. The overall obtained results attest that the proposed cholesterol biosensor can be considered as an alternative tool for practical utilization in real samples.

2. Results and Discussion

2.1. Crystalline, Composition, and Morphological Implications

The powder XRD was employed to describe the crystallography of nanocrystals of Co₃O₄ prepared by the hydrothermal method, as shown in Figure 1. The pure sample of Co₃O₄ is well-characterized by the cubic phase, as shown in Figure 1a, and the diffraction patterns of the sample are well-matched with the standard card no: 01-080-1539. The nanocrystals of Co₃O₄ prepared in the presence of polyethyleneimine were also studied by the XRD technique, and the observed diffraction patterns are enclosed in Figure 1b. The reflections were slightly shifted towards higher two theta angle which could be assigned to the long chain species of the hydrolyzed product and polyethyleneimine, which might produce stress during the crystal formation. The intensity of XRD diffraction patterns indicates the high quality of the nanocrystals of Co₃O₄ prepared in this study. The XRD study has not shown any other phase or impurity in the prepared Co₃O₄. The chemical composition in terms of elements was also studied by EDS, confirming the presence of only Co and O as the main elements in the prepared samples of Co₃O₄, as shown in Figure 2a,b. The EDS and XRD results reveal the pure phase of the prepared nanostructures of Co₃O₄.

The morphology of pure Co₃O₄ and the polyethyleneimine-assisted Co₃O₄ was investigated by SEM, as shown in Figure 3. The pure Co₃O₄ is defined by the self-assembly of big particles and shaping of a platelet-like structure, as shown in Figure 3a,b. The size of the particles is around 200 nm and the length of the platelets is seen as several microns. An addition of polyethyleneimine dispersion in ethanol in the precursor of cobalt oxide has shortened the size of particles, and the morphology of Co₃O₄ seems close to the nanocrystals, as shown in Figure 3c,d. In principle, the long chains of hydrocarbon along with amine functional groups play a vital role in driving the crystal orientation of cobalt oxide, and, consequently, well-controlled cobalt oxide nanocrystals are obtained. The possible role of polyethylenimine might be the provision of additional nucleates which arranged the crystal to grow in the shape of a nanocrystal. Similarly, the particles of Co₃O₄ are well-controlled using polyethyleneimine, due to the slowdown of growth kinetics, and enable the crystal to grow slowly and properly, as shown in Figure 3c,d. The nanocrystals of Co₃O₄ are associated with a large surface area and make better contact with the electrode, thus generating a strong output potential response. The large surface area allows the cholesterol oxidase to be heavily hosted by the Co₃O₄ nanocrystals through strong electrostatic interaction, thus proving itself as a sensitive transducer for the generation of output signal and strongly favoring the catalytic reaction of cholesterol oxidase towards the oxidation of cholesterol.

2.2. Potentiometric Performance of Cholesterol Oxidase-Immobilized Co₃O₄ Nanocrystals towards Cholesterol Determination

The presented cholesterol biosensor based on cholesterol oxidase-immobilized Co₃O₄ nanocrystals was evaluated through the potentiometric method in 10 mM phosphate buffer solution of pH 7.3 [34]. The performance of cholesterol oxidase-immobilized Co₃O₄ nanocrystals was compared with the pristine Co₃O₄ nanostructures, as shown in Figure 4a,b. It can be seen from Figure 4a that the biosensing performance of cholesterol oxidase-immobilized Co₃O₄ platelets is poor in terms of accuracy and precision, suggesting a poorly designed analytical device. However, the cholesterol oxidase-immobilized Co₃O₄ nanocrystals were found highly efficient towards the detection of cholesterol in terms of a wide linear range of 0.005 mM to 3 mM and low limit of detection of 0.001 mM, as shown in Figure 4b. Similarly, the analytical behavior of the proposed method is accurate, as signified by the regression coefficient of 0.99. Additionally, the cholesterol oxidase-immobilized Co₃O₄ nanocrystals have exhibited a Nernstian response, as confirmed by the sensitivity value of 60 mVdec⁻¹. Furthermore, the successive addition method was used to develop the linear range from 0.001 mM to 3 mM for the cholesterol oxidase-immobilized Co₃O₄ nanocrystals, as shown in Figure 5.

This study verifies that the presented cholesterol biosensor has high sensitivity and stability towards the oxidation of the cholesterol substrate during measurement. It strengthened the results presented in Figure 4b. The presented cholesterol is well-characterized by the wide linear range and an excellent sensitivity, which are assigned to its large surface area and excellent co-catalytic properties possessed by the Co₃O₄ nanocrystals. The Co₃O₄ nanocrystals exposed the unique surface for the immobilization of cholesterol oxidase and promoted the activity of the enzyme being a co-catalyst. The enzymatic reaction processing on the cholesterol oxidase-immobilized Co₃O₄ nanocrystals could be described by two different mechanisms. First, the mechanism is followed by the selective activity of cholesterol oxidase towards cholesterol substrate. Second, the mechanism is involved with the cholesterol and cholesterol oxidase-immobilized Co₃O₄ nanocrystals, which have more favorable kinetics than the first reaction mechanism [35]. The superior performance of cholesterol oxidase-immobilized Co₃O₄ nanocrystals could be indexed to synergetic effects [36]. Similarly, the hydrolysis of polyethyleneimine in the organic and inorganic solvents may create the defects and electrical charges [37,38]. The cholesterol reaction with cholesterol oxidase-immobilized Co₃O₄ nanocrystals produced the following substances [39].

Cholesterol + O₂ → 5-3-ketosteroid +H₂O₂

(2)

The product 5-3-ketosteroid is short-lived and transformed into 4-3-ketosteroid due to the isomerism at the trans double bond of the 5-6 steroid ring through an intermolecular shift of 4-6 β sites.

5-3-ketosteroid (isomerisation) → 4-3-ketosteroid

(3)

The potentiometric signal of the presented biosensor is attributed to the reaction mechanism described herein which enabled the generation of charged species floating on the surface of cholesterol oxidase-immobilized Co₃O₄ nanocrystals. The performance of the proposed method is describing the enhanced co-catalytic properties of Co₃O₄ nanocrystals for the cholesterol oxidase by providing the fast response and direct electron transfer kinetics of different catalytic sites between cholesterol oxidase and Co₃O₄ nanocrystals. The inter-electrode study of ten cholesterol oxidase-immobilized Co₃O₄ nanocrystals electrodes was studied in 0.5 mM of cholesterol, as shown in Figure 6.

The potentiometric response of each independent electrode was highly reproducible with less than 5% deviation, indicating excellent activity of cholesterol oxidase-immobilized Co₃O₄ nanocrystals. This study was very vital to fabricate the practical cholesterol-oxidase-immobilized-Co₃O₄-nanocrystals-based configurations at a large scale. Similarly, the repeatability of the same cholesterol oxidase-immobilized Co₃O₄ nanocrystals electrode was investigated by conducting the three repeating measurements of cholesterol at alternative days, in terms of linear range and limit of detection, as shown in Figure 7.

The repeatability experiment has verified the results reported in Figure 4b, revealing the outstanding performance of cholesterol oxidase-immobilized Co₃O₄ nanocrystals due to the strong and favorable microenvironment for the cholesterol oxidase to retain activity for long-term use. The storage life of the cholesterol oxidase-immobilized Co₃O₄ nanocrystals was also evaluated for the period of three weeks, as given in

Table 1. Storage life of cholesterol oxidase-immobilized Co₃O₄ nanocrystals.

No. of Weeks	Linear Range (mM)	Slope (mVdec−1)	Limit of Detection (mM)
1	0.005–3	60	0.001
2	0.004–3	60	0.003
3	0.006–3	60	0.002

. The storage study was conducted, and we have observed that the sensor is applicable even after the period of 3 weeks. The repeatability test was also performed, and both experiments suggested that there is less likely of a chance of a fouling of the sensor. At the same time, we believe that the proper storage after use can avoid the fouling of the sensor.

It is clear that a slight variation in the limit of detection was noticed; however, the linear range and sensitivity of that proposed were preserved. The storage life is related to the environmental conditions in addition to the composition of the electrode and effectiveness of enzyme immobilization. In our study, we kept the modified electrode at 4 °C in a phosphate buffer solution after use. The dynamic response of presented cholesterol oxidase-immobilized Co₃O₄ nanocrystals was also studied in 0.5 mM cholesterol, as shown in Figure 8. The biosensor has shown a fast response time of 1–3 s, suggesting the sensitivity of the cholesterol substrate towards the cholesterol oxidase-immobilized Co₃O₄ nanocrystals. The selectivity of the present cholesterol biosensor was monitored under the common interfering species, as depicted in Figure 9.

The selected interfering species were urea, dopamine, fructose, ascorbic acid, uric acid, and glucose and they were used with 0.05 mM concentration. The output potential signal was negligibly altered in addition to the 0.01 mM and 0.05 mM cholesterol solutions, as shown in Figure 9. However, the same concentration of 0.1 mM of cholesterol has produced output potential by interacting with cholesterol oxidase-immobilized Co₃O₄ nanocrystals, as shown in Figure 9, confirming an excellent selectivity of the proposed method. The analytical features of cholesterol oxidase-immobilized Co₃O₄ nanocrystals towards the detection of cholesterol were monitored by the % recovery method, as given in

Table 2. Percent recovery response of cholesterol oxidase-immobilized Co₃O₄ nanocrystals.

Experiment No.	Added (mM)	Found (mM)	% Recovery
1	1	1.02	102
2	2	1.97	98.5
3	3	2.99	99.6

. The estimated performance has shown an excellent performance of the presented cholesterol biosensor and enables it to be considered as a potential modified electrode for practical aspects.

The fitted circuit for measuring the impedance values of different materials is enclosed, as inset in Figure 10, describing the distinctive Nyquist plots. The impedance study confirms the role of nanocrystals with fast charge transports compared to the platelets structure; however, a slight increase in impedance was noticed due to the insulating nature of cholesterol oxidase and glutaraldehyde. The obtained impedance behavior and values are closely related to reported works [40,41]. The estimated impedance values from simulated EIS data are given in

Table 3. Impedance values from EIS for various presented materials in this study.

Materials	Charge Transfer (Rct)
Pure Co3O4 platelets	4.1 × 103 Ohms
Pure Co3O4 platelets + Cholesterol oxidase	7.8 × 103 Ohms
Co3O4 nanocrystals	1.6 × 103 Ohms
Co3O4 nanocrystals + Cholesterol oxidase Enzyme	2.1 × 103 Ohms

. It is obvious from these values that cholesterol oxidase-immobilized Co₃O₄ nanocrystals confirmed an excellent charge transfer for increasing the performance of the proposed cholesterol biosensor. The performance evaluation of the cholesterol-oxidase-immobilized-Co₃O₄ -nanocrystals-based cholesterol biosensor was compared with the well-established methods using an enzymatic approach in the existing literature, as given in

Table 4. Comparative analysis of cholesterol oxidase-immobilized Co₃O₄ nanocrystals with reported works.

Electrode Material	Experimental Method	Linear Range (mM)	Limit of Detection (mM)	Sensitivity (mVdec−1)	Response Time (s)
ChOx/Co3O4 nanowires/Au [42]	Potentiomtirc	1 × 10−7–1 × 10−3	0.035 × 10−7	−94.031	10
ChOx/ iron nanoparticales [15]	Spectrophotometric	1.3 × 10−3–5.2 × 10−3
ChOx / ZnO [16]	Electrochemical	0.65 × 10−3–10.34 × 10−3
ChOx/ cobalt oxide nanomaterials [17]	Flow injection analysis	4.2 × 10−6–50 × 10−6	4.2 × 10−6	43.5 nA Mm−1 cm−2	15
ChOx/ chitosan-tin oxide nanobiocomposite [18]	Amperomtric	0.26 × 10−3–10.36 × 10−3	0.13 × 10−3	34.7 mA/mg dL −1 cm2	5
ChOx/Fe3O4 nanoparticles [19]	Amperomtric	1.3 × 10−3–5.2 × 10 – 3	0.5 × 10−3		50
ChOx/Co3O4 nanocrystals [Present work]	Potentiometric	5 × 10−6–3 × 10−2	1 × 10−6	60 mV/decade	Less than 1 s

. It is obvious from Table 4 that those different materials have been used for cholesterol detection [15,16,17,18,19]. The presented approach based on cholesterol oxidase-immobilized Co₃O₄ nanocrystals is simple, accurate, sensitive, and cost-effective and more likely has high potential to quantify the cholesterol under the biological matrix. Beside this, the sensor has limitations for the cellular level monitoring of cholesterol in the current form. Therefore, new techniques are required to integrate the proposed material and to quantify the cholesterol levels regularly at the cellular level in order to avoid cardiovascular diseases.

3. Materials and Methods

3.1. Used Chemical Reagents

Urea, cholesterol oxidase derived from Streptomyces sp. (≥20 units/mg), cholesterol, cobalt chloride hexahydrate, polyethyleneimine, d-(+)-Glucose monohydrate (99.5%), l-ascorbic acid (99%), uric acid (99%), ethanol (99.8%), and 1-propanol (99.9%) were received from supplier of Sigma Aldrich at Karachi, Sindh, Pakistan.. The deionized was employed as a solvent medium. The stock solution of cholesterol was made in 10 mM phosphate buffer solution of pH 7.3 by mixing certain amounts of disodium hydrogen phosphate (Na₂HPO₄·7H₂O), potassium dihydrogen phosphate (KH₂PO₄ 99.9%), sodium chloride (NaCl 99.9%), and potassium chloride (KCl 99.0%) in the deionized water. The pH of buffer solution was adjusted with 0.1 M of sodium hydroxide (NaOH 98%) and 0.1 M of hydrochloric acid (HCl 37%). The obtained chemicals were of high analytical grade and used without pretreatment.

3.2. The Preparation of Nanocrystals of Co₃O₄ by Hydrothermal Process

Prior to the synthesis of nanocrystals of Co₃O₄, we fabricated gold-coated glass electrodes, according to our previous study [23]. Briefly, the cleaned glass substrates were kept in Evaporator machine; then, at a high vacuum, the chromium metal of 10 nm was deposited as adhesive layers, followed by the deposition of 100-nanometer thickness of gold layer. Afterwards, the nucleation processing for the deposition of nanocrystals of Co₃O₄ on the cleaned gold-coated substrate was carried out according to the reported work [24]. The precursor solution was prepared by the dissolution of 0.1 M cobalt chloride hexahydrate and 0.1 M urea in 90 mL of deionized water. Then, 50 mg of polyethyleneimine was dispersed in 10 mL of ethanol and sonicated for 20 min until a homogeneous dispersion was achieved. Later, the dispersed solution of polyethyleneimine was added to cobalt precursor. The seed layer-coated gold-coated glass substrate was vertically hung in the beaker by exposing the seeded face towards precursor solution, and the beaker was gently sealed and kept at 95 °C for 4 h in electric oven. After the growth process, a pink product of cobalt hydroxide was deposited on the gold-coated glass substrate and it was washed with deionized water followed by ethanol treatment. The cobalt hydroxide product was transformed into nanocrystals of Co₃O₄ by thermal decomposition at 400 °C for 4 h in air. The pure sample of Co₃O₄ nanostructures was synthesized in the absence of polyethyleneimine by same process. Scheme 1 shows the detailed synthesis process. The crystalline phase of synthesized Co₃O₄ nanostructures was studied by powder X-ray diffraction (XRD) using source of X-rays as CuKα radiation (λ = 1.5418 Å) at 40 kV and 40 mA. The morphology and composition of Co₃O₄ nanostructures were investigated by low-resolution scanning electron microscopy (SEM) at an accelerating voltage of 10 kV and energy dispersive spectroscopy (EDS). The biosensor signal as function of potential was measured via pH meter METTLER TOLEDO, and the dynamic and successive addition stepwise responses were measured through the potentiotat supplied from Netherland. The impedance data were collected through electrochemical impedance spectroscopy using a frequency value from 100 KHz to 10 Hz at sinusoidal potential of 10 mV and zero bias potential in 0.5 mM of cholesterol solution prepared in 10 mM phosphate buffer solution of pH 7.3. The raw impedance data were analyzed through Z-view software with fitted equivalent circuit.

3.3. The Use of Physical Adsorption for the Immobilization of Cholesterol Oxidase onto Nanocrystals of Co₃O₄

The physical adsorption was used for the immobilization of cholesterol oxidase onto nanocrystals of Co₃O₄, as described below. A total of 3 mg of cholesterol oxidase was added to 5 mL of 10 mM phosphate buffer solution of pH 7.3 and 100 µL of cross linker 5% glutaraldehyde was also added. Then, dip coating in the enzyme solution was carried out on the nanocrystals of Co₃O₄ several times, followed by drying at 25 °C. The potentiometric cell set up was based on two electrode configurations using enzyme-immobilized nanocrystals of Co₃O₄ as working electrode and silver–silver chloride (Ag/AgCl) filled with 3.5 M KCl as reference electrode. A 10 mM concentration of cholesterol was prepared in the phosphate buffer solution of pH 7.3 and 1 mL of 5% Triton solution. The triton was used as solubility agent for the cholesterol. Prior to the dissolution of cholesterol into buffer solution, it was solubilized with 2 mL of isopropanol at 55 °C. The dilution method was used to make fresh low-concentration solutions of cholesterol in buffer solution of pH 7.3 before the potentiometric measurements. The desired concentration of interfering species was also prepared in 10 mM phosphate buffer solution of pH 7.3. The output potential experiments of presented cholesterol biosensor were performed at 25 °C.

4. Conclusions

In summary, we have studied the effect of polyethyleneimine hydrolysis in an ethanol/water bi-solvent system on the morphology of Co₃O₄ nanostructures using a hydrothermal method. The prepared Co₃O₄ nanocrystals exhibited a large surface area which was successfully capitalized by the massive loading of the cholesterol oxidase enzyme to fabricate a sensitive and selective potentiometric cholesterol biosensor. The Co₃O₄ nanocrystals have exhibited a cubic crystal geometry and the composition is mainly retained by cobalt and oxygen. The fabricated cholesterol oxidase/ Co₃O₄ nanocrystals were found to be highly efficient in generating the output potential in 10 mM phosphate buffer solution of pH 7.3 using a potentiometric method. Significantly, the cholesterol biosensor displayed a wide linear range of 0.005 to 3 mM and LOD of 0.001 mM. The sensitivity of the biosensor was found to be 60 mVdec⁻¹, indicating an excellent Nernstian response. The fast response of less than 1 s further justifies the efficiency and ultra-sensitivity towards trace levels of cholesterol substrate. The storage life has confirmed the long-term feasibility of the proposed cholesterol biosensor. The percent recovery method was also used to evaluate the practical aspect of the proposed method and it was observed to be satisfactory. Based on the obtained results, we believe that the prepared Co₃O₄ nanocrystals can be of prime consideration for other related electrochemical applications.