Fabrication of Polypyrrole–Polystyrene Sulfonate by Amperometric Biosensor‐Based Electropolymerization for Cholesterol Determination in Serum

ABSTRACT

Since cholesterol triggers many diseases, many methods have been developed for its determination. In this study, an alternative system for cholesterol determination was developed by preparing amperometric biosensors. In the development of the biosensor, Pt/polypyrrole–polystyrene sulfonate film was prepared by electropolymerization of polypyrrole in polystyrene sulfonate medium using platinum surfaces. The cholesterol oxidase enzyme was immobilized on the prepared platinum/polypyrrole–polystyrene sulfonate electrode. For molecular determination in the prepared cholesterol biosensor, a series of enzymatic reactions were performed on the enzyme electrode surface at +0.40 V by utilizing the oxidation of hydrogen peroxide. The effects of environmental conditions such as temperature and pH that affect the performance of the biosensor were investigated, and the most suitable conditions for the biosensor were determined. The linear working range of the amperometric biosensor for cholesterol determination was determined. In enzyme immobilization, calculations were made for the Michaelis–Menten constants K _m and V _max values. Storage life and reproducibility of cholesterol biosensor were determined. Cholesterol determination in biological fluid (blood) was performed with the prepared biosensor. The fact that the fabricated amperometric‐based cholesterol biosensor can be used for the diagnosis of many diseases is important in terms of early diagnosis in the future.

1. Introduction

Enzyme biosensors are the general name given to analytical devices that produce signals proportional to the target analyte concentration. Enzyme biosensors use enzymes and transducers together to produce the signal in question. This signal is formed by the change in proton concentration by the enzyme that catalyzes the reaction, the release or reabsorption of gases such as oxygen or ammonia gas, light emission, absorption or reflector emission, and similar events. The transducer converts this signal into current, potential, temperature change, electrochemical light absorption, and thermal or optical measurable responses [1]. To develop biosensors, various enzymes belonging to the oxidoreductase, hydrolase, and lyase groups are combined with transducers and used in application areas such as health services, pharmaceuticals, the food industry, environmental monitoring, and protection [2, 6].

Polyelectrolytes are polymers (polystyrene sulfonate, polypyrrole, etc.) that have charged polymer chains in polar solvents and can dissociate into small ions with opposite charges, as well as have ionizable groups. The solution properties of polyelectrolytes are quite different from those of simple electrolytes in the presence and absence of excess salt. The reason for this specificity is due to charge interactions. The high charge density in polyelectrolytes produces a strong ionic field. This strong ionic interaction is the source of the characteristic properties of polyelectrolytes [7].

The basis of amperometric systems is based on the current intensities measured in a fixed potential. The signals formed in the system are the determining factor in the speed of mass transfer due to a series of systematic events occurring on the electrode surface. Mass transfers involve the formation of an electroactive product in the environment as a result of biocatalytic reactions or changes in the electroactive substance consumed in the reaction and can be directly monitored with a nonreactive working electrode such as platinum. The second electrode serves as a reference electrode. In the case of an amperometric sensor used as a conducting system, the main difference from the potentiometric sensor is that the signal‐producing species from the products are consumed at the electrode surface. Since the signal‐producing species is consumed at the sensor surface, the product concentration at the interface of the conductor and the bioactive layer is assumed to be zero. For these reasons, the reaction rate at equilibrium in amperometric enzyme sensors is equal to the rate of diffusion of the substrate through the membrane under the condition that the bioactive layer containing the enzyme is surrounded by a semipermeable membrane [8, 9].

Cholesterol is a structural component found in the structure of biological membranes, is essential for life, and is a waxy, fatty substance. It is widely found throughout the body, especially in the brain, nerves, heart, intestines, muscles, and liver. Cholesterol in the body's blood plasma is used as the starting material of some hormones (steroid compounds), vitamin D, and bile acids that digest fats [10, 11]. A very small amount of cholesterol in the blood is sufficient for these processes. High cholesterol levels cause blockage of some blood vessels by accumulating in the walls of these vessels, and thyroid disorder is caused by underactivity of the thyroid gland, a disease caused by degeneration of the kidney tubules, diabetes, and jaundice. Low cholesterol levels lead to a disorder caused by overactivity of the thyroid gland located in the front of the neck and anemia [12, 13]. Cholesterol does not dissolve in the blood under normal conditions. To dissolve in the blood, it must combine with a protein in the liver. This combination of cholesterol and protein is called lipoprotein. High total cholesterol and LDL cholesterol in the blood pose a high risk. Low HDL cholesterol is also a risk. Under normal conditions, there is 130–220 mg/100 mL cholesterol in human blood plasma. Two‐thirds of the cholesterol in the human body is esterified with fatty acids, and one‐third is present as sterols [10, 14]. Values above this are known as high cholesterol levels [10]. The level of cholesterol in the blood or serum is a basic parameter in the diagnosis of coronary heart disease, atherosclerosis, and other clinical diseases and in the evaluation of thrombosis risks and myocardial infarction [2]. In light of the above, the determination of this molecule is very important for the diagnosis of cholesterol‐related adverse effects in the body.

The development of enzymatic and nonenzymatic systems has enabled their use in cholesterol determination. Disadvantages of enzymatic systems over nonenzymatic systems include taking too much time, the need for expert researchers, and expensive chemicals. Such disadvantages do not allow fast and reliable determination of automatic serum cholesterol using nonenzymatic methods. Enzymatic methods for cholesterol determination require complex procedures and are costly due to the high cost of enzymes in each analysis. Sensitivity, selectivity, speed, and stability have been improved and costs have been reduced with biosensors designed to determine cholesterol in serum and blood. The application of enzymatic electrodes for biosensors has been achieved by immobilizing the enzyme in suitable matrices. Cholesterol oxidase immobilization has been performed in various matrices: carbon paste, conductive polymers, nanoparticles, sol–gel, self‐assembled layers (SAM), graphite–Teflon composite, and cellulose acetate [14].

The determination of cholesterol and its use for the diagnosis of many diseases has attracted the attention of researchers. In the studies carried out by Muhammed et al. (2013), a platinum/polyaniline–polypyrrole electrode was prepared by electropolymerization of pyrrole and aniline in a sulfuric acid medium. The determination of cholesterol and its use for the diagnosis of many diseases has attracted considerable attention from researchers. They immobilized cholesterol oxidase to this electrode system. Cholesterol determination was made at +0.70 V based on hydrogen peroxide oxidation occurring in the enzymatic reactions on the electrode surface resulting from enzyme immobilization. The effects of environmental parameters such as pH and temperature on the biosensor were determined in the resulting system. They determined the linear working range of the biosensor for cholesterol as 1.8 × 10⁻⁵–5.0 × 10⁻⁵ M. The optimum pH value of the immobilized enzyme was found to be 7.0, and the optimum temperature value was found to be 60°C. The K _m value of the immobilized enzyme was calculated as 3.3 mM and V _max value as 5.26 mM/min. It was observed that the biosensor maintained its activity at 82% after 30 measurements and lost approximately 40% of its initial amperometric response after 23 days. Cholesterol was determined in biological fluid (blood) with the prepared biosensor [15]. Singh et al. [2] used an electrochemical impounding technique to construct an amperometric cholesterol biosensor by immobilizing cholesterol oxidase and cholesterol esterase enzymes together on a conductive polypyrrole film. The electrochemical polymerization process was carried out at 0.8 V with the help of two electrode cells. They experimentally determined the linear working range, temperature, optimum pH value, working potential, and shelf life of the amperometric biosensor for cholesterol. They found the linear working range as 1–8 mM, shelf life as 4 weeks at +4°C, optimum pH as 6.5–7.0, temperature as 45°C, and working potential as 0.50 V. They calculated the sensitivity of the electrode as 0.15 µA/mM and the observed K _m value as 9.8 mM. They determined the conductivity of the polymer film as 3.0 × 10⁻³ S/cm [10]. Katrlik et al. [16] designed a biosensor without interference effect for direct determination of cholesterol. The biosensor system consists of immobilized enzyme peroxidase modified to a planar gold electrode, insoluble mediator, acetate cellulose layers, and cholesterol oxidase enzyme in buffer solution. Cellulose acetate layers and low potential applied reference electrode prevented interferences of ascorbate, urea, lactate, glucose, nitrite, and nitrate in biosensor response and allowed direct cholesterol determination without pretreatment of the sample. Cholesterol was analyzed in linear working range concentrations (1.1–40 µmol/L) in buffer solution. They used the transmission system of the biosensor (without cholesterol oxidase solution) for rapid determination of hydrogen peroxide in buffer solution in the concentration range of 1–250 µmol/L. The delivery system of the biosensor showed good storage time, retaining 77% of the initial activity of the biosensor after 50 days [16]. In their study, Solanki et al. [14] immobilized cholesterol oxidase covalently on polyaniline–polypyrrole copolymer. The electrochemical coating was done on glass indium tin oxide (ITO) layers, and glutaraldehyde was used as a crosslinker. The properties of the films were found using infrared spectroscopy, UV–visible spectroscopy, photometric, amperometric techniques, and scanning electron microscopy (SEM). The linear working range of cholesterol was determined to be 1–10 mM using poly(An‐co‐Py)/ChOx bioelectrode. The highest pH value for cholesterol oxidase activity in poly(An‐co‐Py)/ChOx bioelectrode was found to be pH 7.0, with an optimal temperature of 25°C. The sensitivity of the poly(An‐co‐Py)/ChOx electrode was measured as 93.35 µA/mM and its stability was maintained for 10 weeks at +4°C [8]. Pundir [17] designed an enzyme strip for serum cholesterol determination by co‐immobilizing cholesterol esterase, cholesterol oxidase, and peroxidase enzyme mixtures on polyvinyl chloride (PVC). The kinetic parameters of the prepared enzyme strip were found to be an optimum pH of 7.2, optimum temperature of 45°C, shelf life of 4 months, and repeatability of 200 measurements [17].

It is known that cholesterol is determined by many different methods in detailed literature studies. In this study, unlike the literature, an amperometric‐based PP‐PPS–modified cholesterol biosensor was prepared. This sensor has many advantages as an alternative to cholesterol biosensors found in the literature. The cholesterol biosensor has a wide working range and the ability to go down to low concentrations, high reproducibility, and a good shelf life. Considering all these advantages, the cholesterol biosensor fabricated within the scope of this study has the potential to be used in the diagnosis of many diseases.

2. Materials and Methods

Pyrrole (98%; Aldrich), polystyrene sulfonate (Aldrich), sodium chloride (99.5%; Merck), hydrogen peroxide (30%; Sigma‐Aldrich), uric acid (99%; Sigma), paracetamol (acetaminophen; 99.5%), l‐(+)‐ascorbic acid (99%; Carlo Erba), glucose (Merck), sodium hydroxide (99%; Carlo Erba), Triton X‐100 (Merck), lithium carbonate (45%; Merck), hydrochloric acid (36.5%; BDH), monosodium hydrogen phosphate (99%; Merck), disodium hydrogen phosphate (99%; Merck), cholesterol (Merck), propan‐2‐ol (Merck).

2.1. Coating of Polystyrene Sulfonate and Polypyrrole on Platinum Surface

2.1.1. Cleaning of the Electrode Surface

Before the electrode surface coating process, chemical, mechanical, and electrochemical cleaning processes were performed on the platinum plate used in the study. Before starting the processes, the platinum surface was thoroughly polished with sandpaper (zero number) and then immediately flame‐treated. Platinum plates were cleaned by keeping them in acetone (≥ 99.9), ethyl alcohol (96%), concentrated HCl, and concentrated HNO₃ for 5 min. The platinum plates removed from these solutions were washed with pure water and dried, and the surfaces were cleaned [18]. The above procedures were repeated before each coating process for the cleaning process. The cleaned Pt plate was coated with polypyrrole–polystyrene sulfonate as a polymeric film on the surface of the electrode. The coating of the polypyrrole was done with the triple electrode system by electropolymerization. In the electropolymerization processes, the working electrode was a Pt plate cut to approximately 0.5 cm², the counter electrode was a platinum wire, and the reference electrode was an Ag/AgCl electrode. The pyrrole cell concentration was adjusted to 0.1 M (106 µL pyrrole, 5 mL polystyrene sulfonate, and 4.894 mL pure water) and used to obtain the polypyrrole–polystyrene sulfonate film. The cleaned platinum electrode was immersed in this solution, and argon gas was passed through it for 10 min to remove the oxygen gas in the environment. The voltammetry technique was used in the electropolymerization process. While using the voltammetry technique, the coating process was carried out as a result of 18 transformations with a scanning speed of 20 mV/s at the potentials of −0.8 to +0.8 V. After the polypyrrole–polystyrene sulfonate film coating, the electrode surfaces were washed with deionized water and kept in phosphate buffer [19].

2.2. Determination of the Sensitivity of Platinum/Polypyrrole–Polystyrene Sulfonate Electrode to Hydrogen Peroxide

The previously prepared Pt/polypyrrole–polystyrene sulfonate electrode was immersed in 9 mL of 0.1 M phosphate buffer and 1 mL of 1 M NaCl solution together with the Ag/AgCl reference electrode and platinum wire. The working electrode was allowed to come to equilibrium at a constant potential of 0.4 V, and the equilibrium current was noted. H₂O₂ solutions with intracellular concentrations ranging from 1.0 × 10⁻³ to 1.0 × 10⁻⁷ M were added to the equilibrium electrodes. At the end of each addition, the cell was stirred for 300 s and then waited for 200 s to record the current values. Due to the concentration change that occurred as a result of each addition, the differences between the equilibrium currents (Δi) were calculated, and graphs were obtained according to the H₂O₂ concentration.

2.3. Free Enzyme Study With Pt/PPy‐PSS Film Electrode

The working electrode prepared as stated in the leaning of the electrode surface section was immersed in a solution containing 9 mL of buffer solution and 1 mL of support electrolyte (NaCl) and brought to equilibrium at a constant potential of 0.40 V. Then 100 µL of cholesterol oxidase enzyme was added and allowed to equilibrate in the enzyme medium, and the equilibrium current was recorded. Then cholesterol was added at increasing concentrations between the intracellular concentration of 1.0 × 10⁻⁷–1.0 × 10⁻³ M. After each addition, the reaction was stirred for 300 s to complete, and the current was read 200 s after the end of the stirring. Δi values were calculated for each concentration by taking the differences between the current values read and the equilibrium current.

2.4. Preparation of the Cholesterol Biosensor Immobilization by Entrapment Method

Before enzyme immobilization, the platinum plate was cleaned, as previously mentioned. After cleaning the Pt plate, 4.394 mL of water, 106 µL of pyrrole, 5 mL of polystyrene sulfonate, and 0.5 mL of cholesterol oxidase (ChOx) were added into the cell to prepare a 10 mL cell. Argon gas was passed through the system for 10 min to remove oxygen gas. Then, using the cyclic voltammetry technique, the cholesterol oxidase enzyme was transported to the surface during the electropolymerization of pyrrole on the platinum electrode surface in the polystyrene sulfonate medium at a scanning speed of 20 mV/s between −0.8 and +0.8 V in 18 cycles. The surface of the resulting platinum/polypyrrole–polystyrene sulfonate–cholesterol oxidase (Pt/PPy‐PSS‐ChOx) biosensor was washed with plenty of pure water. The cholesterol sensitivity of the biosensor prepared by the entrapment method was examined and plotted on the graph. The obtained biosensor was stored in the refrigerator at +4°C in phosphate buffer when not in use.

2.5. Determination of Cholesterol Sensitivity of Biosensor

In the biosensors prepared for cholesterol sensitivity, triple electrode systems were preferred. In the electrode system, the working electrode was the biosensor, the reference electrode was the Ag/AgCl electrode, and platinum wire was used as the counter electrode. The electrochemical cell was kept waiting until it reached a constant potential of 0.4 V by adding 9 mL of pH 7.5 phosphate buffer and 1 mL of 1 M NaCl solution, and the equilibrium current was determined. Cholesterol solutions were added to this system at increasing concentrations, mixed for 300 s, and kept waiting for another 200 s, and the currents were noted.

2.6. Determination of Optimal Operating Conditions for the Pt/PPy‐PSS‐ChOx Biosensor

Studies on pH, temperature, substrate concentration, reusability, and shelf life affecting biosensor performance were reviewed in this section. For the effect of pH on the enzyme biosensor, phosphate buffers (0.1 M) were prepared in various pH ranges (pH 6–9). A total of 9 mL of phosphate buffer prepared at different pHs was added into the cell, while 1 mL of NaCl solution was added. The equilibrium current was determined for the biosensor balanced until 0.4 V. After these processes, the phosphate buffer with pH 6 and the cholesterol solution prepared were added at a concentration of 5 × 10⁻⁵ M. The resulting o‐cell medium was stirred for 300 s, the currents at the end of 200 s were recorded, and the differences between the equilibrium currents were determined for pH 7.5. The same processes were performed for both other pH ranges and different temperatures. The most suitable working conditions for pH and temperature were found, as stated above.

To determine the effect of substrate concentration on the prepared biosensor, the biosensor was immersed in a solution containing 9 mL of phosphate buffer (pH 7.5, 0.1 M) and 1 mL of sodium chloride (1 M). When the potential was 0.40 V, the equilibrium current was recorded, and cholesterol was added to the cell at different concentrations (0.1 µM–1 mM). After each addition, the solution was stirred for 300 s, the current at the end of 200 s was read, and the differences between the equilibrium current were determined; Δi values were determined for different cholesterol concentrations. The working range of the biosensor was determined by drawing the Δi graph (Michaelis–Menten curve) for various cholesterol concentrations. In addition, the K _m (observed) and V _max values specific to the cholesterol oxidase enzyme, which are Lineweaver–Burk parameters, were determined from the obtained curves.

To measure the reusability of the biosensor prepared as described above under the best working conditions, consecutive measurements were taken with intracellular cholesterol solution at a concentration of 5.0 × 10⁻⁵ M. The amperometric response current of the biosensor was measured at a fixed cholesterol concentration of 5.0 × 10⁻⁵ M for a total of 13 measurements at different time intervals for 23 days. The biosensor, whose measurement process was completed, was kept in the refrigerator at +4°C in buffer solution until the next measurement process. Δi values were calculated by taking the differences between the equilibrium current and the current value read during each measurement.

2.7. Substances Interfering With the Pt/PPy‐PSS‐ChOx Biosensor

The interference effects of ascorbic acid, paracetamol (acetaminophen), uric acid, and glucose, which are substances present in biological fluids and may have an interference effect, were examined on the amperometric response current obtained at a concentration of 1.0 × 10⁻⁵ M cholesterol under the best operating conditions of the biosensor prepared. In this study, the concentrations of the interfering species were taken to be the same as those found in blood.

To determine the interference of ascorbic acid in Pt/PPy‐PSS‐ChOx biosensor, 1 × 10⁻⁴ M ascorbic acid was added to the cell solution prepared as described in the previous sections and stirred for 300 s, and the currents at the end of 200 s were recorded. After measuring both ascorbic acid‐ and cholesterol‐induced response current, 1 × 10⁻⁵ M cholesterol response current was subtracted, and ascorbic acid response current was found. The total response current was compared to the response current of ascorbic acid, and the percentage interference caused by ascorbic acid was found. The same procedures were performed for paracetamol, glucose, and uric acid at 1 × 10⁻⁴ M concentration, and the percentage interference values are presented in Table 1.

TABLE 1.

Substances interfering on the biosensor (at a concentration of 1 × 10⁻⁴ M cholesterol) and their interference effects.

Interfering substances	Working concentrations (M)	The ratio of the response current of the interfering substance to the total current (%)
Glucose	1 × 10−2	49.26
Glucose	5 × 10−3	50.37
Uric acid	1 × 10−6	21.15
Uric acid	1 × 10−5	32.79
Uric acid	1 × 10−4	50.22
Ascorbic acid	1 × 10−6	11.86
Ascorbic acid	1 × 10−5	24.93
Ascorbic acid	1 × 10−4	43.53
Paracetamol	1 × 10−6	28.77
Paracetamol	1 × 10−5	54.83
Paracetamol	1 × 10−4	19.07

2.8. Determination of Cholesterol in Biological Fluid (Blood)

Samples were taken from three different people to determine the prepared biosensor in the biological fluid blood. Sample collection procedures were performed on healthy individuals. These samples were placed in sterile containers and stored in the refrigerator until analysis. Dilution procedures were performed to determine cholesterol in blood serum. Thanks to the dilution procedure, the cholesterol concentration in the blood serum was brought to the linear working range of the biosensor, and the interfering substances were reduced to a concentration below the determination limit of the biosensor. The biosensor, which was prepared and brought to the above‐mentioned, was placed in the electrochemical cell with 9 mL of phosphate buffer solution with a pH of 7.5 and 1 mL of sodium chloride solution to determine the total cholesterol amount in the blood. The balance current was recorded by bringing it to balance at an electrode potential of 0.40 V. Certain volumes were taken from the blood serum and added to the cell where the biosensor was located in a way that there would be a dilution process of 200 times. Then, a constant potential of 0.40 V was applied, and the current passing through the cell was recorded. Then, cholesterol solutions of known concentration were added, and the current values were recorded. The Δi value was determined from the difference between each equilibrium current and the current read. The same procedures were performed for the three blood samples taken. Cholesterol concentrations were determined by the standard addition method.

2.9. Characterization Techniques

SEM, atomic force microscope (AFM), and surface water contact angle measurements were performed to determine the morphological analysis of the film electrode and the change in the chemical composition on its surface.

3. Results and Discussions

In this study, conductive polymers such as polypyrrole and polystyrene sulfonate were coated on the platinum surface. Pyrrole and polystyrene sulfonate were deposited on the electrode surface by electropolymerization method on this prepared film, while the cholesterol oxidase enzyme was immobilized in the polymer by entrapment method. Analytical and biochemical properties of the prepared biosensor were investigated. pH, temperature, substrate concentration, and interference effects on the biosensor were investigated. In addition, the repeatability and shelf life of the biosensor were determined. H₂O₂ is released as a reaction product in some of the enzymatic reactions. Most of the prepared biosensors are based on the principle of oxidation of H₂O₂ formed as a result of enzymatic reaction on the surface of the working electrode at the applied constant potential [3, 4, 5, 20, 21, 22]. H₂O₂ is formed according to the following reaction:

$$\mathrm{Cholesterol}\mathrm{esters}+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{Cholesterol}\mathrm{esterase}}{\to }\mathrm{Cholesterol}+\mathrm{fatty}\mathrm{acid}$$

$$\mathrm{Cholesterol}+{\mathrm{O}}_2\stackrel{\mathrm{Cholesterol}\mathrm{oxidase}}{\to }\mathrm{Cholest}-4-\mathrm{en}-3-\mathrm{on}+{\mathrm{H}}_2\mathrm{O}$$

The hydrogen peroxide formed is oxidized at a constant potential on the platinum electrode surface according to the following reaction:

$${\mathrm{H}}_2{\mathrm{O}}_2\to {\mathrm{O}}_2+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}$$

When working at a potential where oxidation will occur, the anodic current passing through the circuit is proportional to the concentration of hydrogen peroxide formed, and therefore, to the cholesterol concentration. Although the principle of this study is the same as the principle above, no previous study has been found on this film and cholesterol oxidase enzyme. Hydrogen peroxide is formed as a result of the enzymatic reaction between the Pt/PPy‐PSS‐ChOx biosensor and cholesterol.

The cholesterol oxidase enzyme converts cholesterol to cholest‐4‐en‐3‐one, and as a result of the reaction, hydrogen peroxide is released. The Pt/PPy‐PSS‐ChOx biosensor was created by oxidizing the hydrogen peroxide, which is the product of this reaction. Therefore, the sensitivity of the prepared Pt/PPy‐PSS electrode to H₂O₂ was examined, as explained in the relevant section. The Δi values generated by the amounts of hydrogen peroxide formed were plotted on the graph. It was found that the graph changed linearly with the amount of hydrogen peroxide released, and the value of the regression coefficient was R ² = 0.997. This showed that the prepared biosensor was highly sensitive to hydrogen peroxide (Figure 1).

FIGURE 1.

Sensitivity of Pt/PPy‐PSS electrode to hydrogen peroxide.

3.1. Investigation of Cholesterol Sensitivity of Pt/PPy‐PSS Film Electrode in Free Enzyme Environment

It was observed that the anodic current, formed as a result of the reaction between the free enzyme with its substrate, cholesterol, in the prepared cell, as mentioned in the experimental section, increased with the rise in cholesterol concentration due to diffusion of H₂O₂ to the electrode surface. The obtained Δi values were plotted (Figure 2).

FIGURE 2.

Sensitivity of Pt/PPy‐PSS film electrode to cholesterol in free enzyme medium.

The electrochemical detection of hydrogen peroxide, which is produced as a result of the enzymatic reaction caused by cholesterol oxidase, can be explained by the fact that the response currents are parallel to the cholesterol concentration.

3.2. Immobilization With Entrapment Method

This study aimed to select the appropriate immobilization technique for the cholesterol oxidase enzyme. For this purpose, a biosensor was prepared using the immobilization method as described in the experimental section, and amperometric response currents were recorded as a result of increasing cholesterol additions. The Δi values obtained for the immobilization technique with the immobilization method were recorded (Figure 3).

FIGURE 3.

Immobilization by entrapment (effect of cholesterol concentration on the amperometric response, 0.1 M phosphate buffer pH 7.5, 25°C).

As seen in the graph, high currents were obtained when immobilization was performed using the entrapment method. In addition, the reusability of the biosensor was measured (Figure 4).

FIGURE 4.

Repeatability (with the entrapment method,0.1 M phosphate buffer, pH 7.5, 25°C).

It is seen that the biosensor prepared with the capture method retains approximately 75% of its initial activity after 23 measurements.

SEM was used to obtain information about the morphology of the film electrode with the Pt/PPy‐PSS andPt/PPy‐PSS‐ChOx biosensor (Figure 5a, b).

FIGURE 5.

(a) SEM photo of Pt/PPy‐PSS film and (b) SEM photo of Pt/PPy‐PSS‐ChOx biosensor.

When the SEM photographs of the electrodes with and without enzymes are examined, it is seen that the two are different from each other. The natural appearance of the PPy film is granular. When the magnification is increased and SEM is taken, it is seen that this structure resembles the cauliflower structure [23]. In addition, as the dopant amount increases, the particle size of PPy increases, too. The granular structure is more compact and has lower pores due to the increasing dopant concentration. As a result of the immobilization of cholesterol oxidation enzymes, it was observed that the structure of the film electrodes in the cauliflower structure changed. Morphological analysis shows that the enzyme was successfully immobilized on the Pt/PPy‐PSS film.

In addition to investigating the effectiveness of the enzyme biosensor, the composition (hydrophilic and hydrophobic characters) and morphology of the surface should be well‐defined. Especially, in defining biochemical reactions and carrying out these reactions homogeneously, hydrophilic, and hydrophobic interactions may be directly related. Therefore, water contact angle measurements were taken to determine the chemical composition of the surfaces of enzyme biosensors. It is seen in Figure 6 that the film formed as a result of enzyme immobilization has a more hydrophilic character compared to the other film. The water contact angles were measured to be 80° for the PPy‐PSS film electrode and 22° for the Py‐PSS‐ChOx biosensor. The result is consistent with the expectation of a hydrophilic character due to the proteinic structure of the enzymes [24, 25]. Obtaining more hydrophilic surfaces with enzyme immobilization shows that the immobilization process was carried out successfully.

FIGURE 6.

(a) AFM and contact angle photograph of Pt/PPy‐PSS film and (b) AFM and contact angle photograph of Pt/PPy‐PSS‐ChOx biosensor.

AFM images taken for changes in morphological processes are given in Figure 6. To specifically obtain information about the roughness, the RMS value was measured to be 204 nm on the surface with the enzyme and 286 nm on the surface without the enzyme. With the immobilization of cholesterol oxidase, the roughness value of the surface decreased, and therefore, it was understood that the enzyme biosensor had a more regular morphological structure.

3.3. Determination of Optimal Operating Conditions for the Pt/PPy‐PSS‐ChOx Biosensor

One of the most important factors affecting the activity of biosensors is pH because the activity of enzymes changes depending on pH. To determine the best working pH of the biosensor, the amperometric response currents obtained as a result of the experiments conducted with the prepared biosensor according to the experimental section were plotted against pH (Figure 7).

FIGURE 7.

Role of pH in biosensor activation (0.1 M phosphate buffer, 25°C).

When the graph is examined, the currents increased up to pH 7.5, and the maximum response current was observed at pH 7.5. The response currents started to decrease after pH 7.5. The increase in response currents is expected as the best pH value is approached. Since enzyme and cholesterol molecules are acidic and basic groups, (E–S) activated complexes must be formed most easily; that is, these groups must be in a certain ionization state for the reaction rate to be maximum. In other ionizations, the formation of the (E–S) complex will be difficult, and the reaction rate will decrease, so the decrease in currents is an expected situation since the activity of the enzyme decreases as it moves away from the best working pH value [26]. In the literature, pH values for cholesterol oxidase immobilized as single or double on different support materials were found to be 7.0, 7.25, 7.4, 7.0, 6.5–7.5 [2, 15, 27, 28, 29].

Temperature is an important factor affecting the stability of enzymes and the speed of enzymatic reactions. Each enzyme has a temperature at which it shows maximum activity [1]. Therefore, it is important to examine how the response of the biosensor we prepared changes with temperature. To determine the best operating temperature of the biosensor, the amperometric response currents obtained as a result of the experiments conducted with the prepared biosensor according to the experimental part were plotted against temperature (Figure 8).

FIGURE 8.

The role of temperature in biosensor effectiveness (0.1 M phosphate buffer, pH 7.5, 25°C).

When the graph is examined, it is seen that the enzyme shows low activity at low‐temperature values, and the amperometric response currents increase as the temperature increases. It was observed that the maximum response current was at 40°C. It is seen that the activity decreases after 40°C and almost disappears after 55°C. In the literature, temperatures such as 60°C, 35°C, 49°C, and 37°C were found for cholesterol oxidase enzyme immobilized on different support materials [14, 28, 30, 31]. As the temperature increases, the protein structure of the enzymes undergoes a denaturation process, and they lose their activity at a high rate [26]. The protein structure of the enzyme may deteriorate in long‐term studies at 40°C. Therefore, in this study, room temperature, where enzyme activity is preserved, was chosen as the optimum temperature.

In an environment where the enzyme concentration and all other conditions are constant, the rate of the enzymatic reaction initially shows a linear increase with increasing substrate concentration; however, as the substrate is added, the rate gradually increases less due to the filling of the active center of the enzyme and remains constant at a certain level. As explained in the experimental part, measurements were taken with the Pt/PPy‐PSS‐ChOx biosensor at an electrode potential of 0.40 V until the substrate saturation of the enzyme. When the Michaelis–Menten graph (Figure 4) drawn according to the obtained results is examined, the response currents started to remain constant after the addition of 1.0 × 10⁻⁴ M cholesterol.

It is difficult to determine the characteristic properties with the Michaelis–Menten equation, which is the equation of a hyperbolic curve. In this case, the Lineweaver–Burk graph obtained by making the necessary adjustments to the equation is given in Figure 9. Using the Lineweaver–Burk graph, the K _m (observed) value for the single‐electrode system was calculated as 1.17 × 10⁻⁴ mM and V _max (observed) as 0.39 µA/min. In the literature the K _m values for cholesterol oxidase enzyme immobilized on different support materials and purified from different sources were found to be 3.3, 1.16, and 7.9 mM, and V _max was found to be 5.26, 1.78, mM/min [9, 15, 21, 22, 27].

FIGURE 9.

The Lineweaver–Burk graph drawn for the Pt/PPy‐PSS‐ChOx biosensor (0.1 M phosphate buffer, pH 7.5, 25°C).

The relationship between the reaction rate of a given amount of enzyme and the substrate concentration is initially linear but later takes on a hyperbolic shape. In the range where the relationship is linear, the enzyme exhibits first‐order kinetics. The lowest and highest concentration values in this range determine the working range of the biosensor. It was understood that the response currents obtained from the prepared biosensor showed a linear change in cholesterol concentrations, and quantitative analyses could be performed, especially in these concentration ranges of 5.0 × 10⁻⁶–1.0 × 10⁻⁴ M (Figure 10).

FIGURE 10.

Calibration curve of the Pt/PPy‐PSS‐ChOx biosensor.

3.4. Determination of Reusability of Biosensor

In the graph drawn for the repeatability of the Pt/PPy‐PSS‐ChOx biosensor, decreases were seen in the response currents with the increase in the number of measurements (Figure 5). The decrease in enzyme activity over time can be considered as the main reason for the decrease in currents. As a result of 23 measurements made in the Pt/PPy‐PSS‐ChOx biosensor, the enzyme activity was preserved at a rate of 75%. This situation shows that its activity is still preserved at a high rate, and the immobilization technique is suitable for the enzyme. This high repeatability shows that the biosensor can perform consecutive analyses. The high cost of enzyme kits used in a single measurement for routine analyses makes the biosensor we prepared advantageous in this sense. Our literature research also shows that the prepared biosensor will be an alternative to those in the literature and has high repeatability [15, 27].

3.5. Determination of Shelf Life of the Pt/PPy‐PSS‐ChOx Biosensor

In the studies conducted for the shelf life of the Pt/PPy‐PSS‐ChOx biosensor, the change in amperometric response currents with the number of days was investigated (Figure 11). In the obtained data, it was observed that the biosensor lost 45% of its activity after 23 days and maintained its initial activity at 55%. In particular, changes that occur as a result of the interaction of the active center of the enzyme with the temperature of the environment, air quality, and chemical substances may cause a decrease in biosensor activity. In the literature, there are some biosensors that maintain their stability in the first 10 days and the first 6 weeks, as well as biosensors that lose 40% of their activity in 23 days and 73% of their activity in 78 days [15, 27].

FIGURE 11.

Investigation of shelf life of the Pt/PPy‐PSS‐ChOx biosensor (0.1 M phosphate buffer, pH 7.5, 25°C).

3.6. Substances Interfering on the Biosensor

In biosensor applications, it is necessary to investigate the interfering substances in the studies conducted on real samples. In this context, the substances that can cause interference in the Pt/PPy‐PSS‐ChOx biosensor that we prepared (ascorbic acid, paracetamol, glucose, and uric acid) and the information with the amperometric response currents are presented in Table 1.

The amperometric response current ratios in the Pt/PPy‐PSS‐ChOx biosensor for 1 × 10⁻⁴ M concentrations of uric acid, ascorbic acid, paracetamol, and glucose were found to be 50.22%, 43.53%, 54.83%, and 50.37%, respectively. Although these high ratios pose problems in terms of the usability of the biosensor, interferences can be reduced to lower levels with additional dilution procedures [20, 32–35].

3.7. Determination of Cholesterol in Biological Fluid (Blood)

The concentration values corresponding to the measured amperometric response currents at the end of the cholesterol analysis in biological fluid were calculated by the standard addition method, and the obtained results are given in Table 2. Table 1 shows the results of the substances that can interfere with cholesterol in the study. These results show that the interference of the interfering compounds has not been completely eliminated. As a result of the interference of these compounds, which can also be found in the serum, the cholesterol results were different from the hospital results.

TABLE 2.

Cholesterol concentration calculated in blood samples.

Blood numbers	The Pt/PPy‐PSS‐ChOx biosensor results (mg/dL)	Hospital results (enzyme‐based spectrophotometric method) (mg/dL)
1	255	195
2	305	268
3	297	278

In a study conducted in 2005, Tan et al. determined that the amount of cholesterol in blood samples of a cholesterol biosensor prepared using multi‐walled carbon nanotubes was very close to the amount added [36]. Later, in another study conducted in 2016, Dervisevic et al. determined that the amount of cholesterol in blood samples was slightly higher than the amount of cholesterol added to the system they prepared for the cholesterol biosensor [37]. Bui et al. prepared a cholesterol biosensor in 2016 and determined that the amount of cholesterol in blood samples was high [38]. In 2022, Wu et al. found that cholesterol was high in the biosensor they developed for the determination of cholesterol in blood [39]. In the studies conducted to date, different strategies have been developed for the analysis of cholesterol in blood. The common feature of these strategies is that the cholesterol values, together with the interfering substances, were higher than the amount added to the system. The results obtained in our study are consistent with the results of the studies conducted to date and show that the prepared cholesterol biosensor has the potential to be used for blood samples.

4. Conclusions

The Pt/PPy‐PSS‐ChOx biosensor linear working range was found to be 5.0 × 10⁻⁶–1.0 × 10⁻⁴ M. Cholesterol determination in biological fluid (blood) was carried out in the working range of 5.0 × 10⁻⁶–1.0 × 10⁻⁴ M. The best working pH was found to be 7.5. The best working temperature was found to be 40°C. Reusability was investigated (at the end of 23 measurements, it was observed that the biosensor preserved its activity by 75%). When compared to the literature values, it is seen that the reproducibility of the biosensor prepared is satisfactory. Shelf life was investigated (at the end of 23 days, it was observed that it preserved approximately 55% of the initial amperometric response). When compared to the literature values, it is seen that the shelf life of the biosensor prepared is satisfactory. For the prepared Pt/PPy‐PSS‐ChOx biosensor, K _m (observed) value was found to be 0.046 mM, and I _max (observed) was found to be 0.31 µA. As a result, the biosensor prepared has a wide working range and the ability to reach low concentrations, high reproducibility, and a good shelf life.

Author Contributions

Conceptualization: Alaa Anwer Ali Dada and Servet Çete. Methodology: Alaa Anwer Ali Dada, Ertan Yildirim, Sinan Mithat Muhammed, and Servet Çete. Investigation: Alaa Anwer Ali Dada, Ertan Yildirim, and Servet Çete. Writing – original draft: Ertan Yildirim, Sinan Mithat Muhammed, and Servet Çete. Final writing and editing: Ertan Yildirim, Sinan Mithat Muhammed, and Servet Çete. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Dada A. A. A., Yildirim E., Muhammed S. M., and Çete S., “Fabrication of Polypyrrole–Polystyrene Sulfonate by Amperometric Biosensor‐Based Electropolymerization for Cholesterol Determination in Serum.” Biotechnology and Applied Biochemistry 72, no. 5 (2025): 1330–1340. 10.1002/bab.2742

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.