Measuring the presence of cholesterol in foods has been the subject of extensive research. In general, the production of electrodes for cholesterol detection is of great importance in terms of clinical tests. Additionally, monitoring the cholesterol level in blood and food is a critical parameter for diagnosing and preventing many diseases ^[1][34]. Researchers have thus far developed different methods to monitor cholesterol, including colorimetry ^[2][3][4,35]. These are chromatographic ^[4][5][6][7][36,37,38,39], fluorometric ^[7][8][39,40], and chemiluminescent methods ^[9][41]. In general, many of these mentioned methods require expensive instrumentation and complex preparation procedures to precipitate lipoproteins, or lack an acceptable sensitivity and selectivity. Various colorimetric, polarographic, chromatographic, spectrophotometric, and biosensor methods are used in the determination of cholesterol. However, these are generally time-consuming and expensive systems. Measurements made via gas and liquid chromatography are the most suitable for cholesterol detection in terms of separating cholesterol from other similar compounds as well as determining its quantity ^[10][42]. Therefore, it is of great importance to develop systems that can determine the amount of cholesterol more accurately, in a shorter time, and at a lower cost. Generally, conventional methods such as colorimetry, spectrophotometry, fluorimetry, polarography, thin layer chromatography, gas chromatography, and high-performance liquid chromatography are used to measure cholesterol levels in the sample under investigation ^[11][12][43,44]. The majority of current sensors used are capable of adequately detecting free or esterified cholesterol. However, their originality is poor, complex, expensive, labor-intensive, and time-consuming ^[13][22]. In the last decade, enzymatic, non-enzymatic, and redox mediator-based sensors have been developed for cholesterol detection in enzymatic systems, enzymes such as cholesterol oxidase (ChOx) or cholesterol esterase (CE) catalyze the hydrolysis of cholesterol ester, resulting in the formation of fatty acids and free cholesterol. Because of the complexity of the matrix, chromatographic measurements with different types of detectors are the most common methods for cholesterol determination ^[14][15][45,46]. The main advantages of chromatographic methods are high selectivity, low LOD (depending on the detection used), and high accuracy. On the other hand, these techniques traditionally have disadvantages such as high cost and high personnel requirements ^[16][17][47,48]. Sensors that can only measure cations and anions with the classical electrochemistry system have enabled the determination of many substances with the inclusion of a biomaterial in the system. Biosensors find usage in applications such as bacteria and virus determination, agriculture, veterinary, biomedical sector, toxic gas analysis in mining enterprises, food production and analysis, drug analysis, military applications, process control, environmental protection and pollution control, clinical diagnosis, bioreactor control, agriculture veterinary, and industrial waste ^[18][19][20][49,50,51]. With the developing technology, biosensors, especially enzymatic biosensors, are used in hospitals and the food industry. Biosensors can be used for complex parameters such as the detection of foreign substances in foods, freshness, and aroma control ^[21][22][23][52,53,54]. In addition to this, biosensors can be used in the fight against drugs and preventing the misuse of drugs ^[24][55]. Although biosensors have high specificity and speed, they require the development of an enzyme stabilization method. This is one of the main disadvantages of this type of system. The instability of the enzyme poses a problem in obtaining accurate and precise results ^[25][56]. Proper orientation and high surface biocompatibility of the enzyme play an important role in facilitating electron transfer between the enzyme and the electrode surface. In these sensors, random orientation may lead to a decrease in the concentration of active enzymes on the electrode surface, which may result in a decrease in the sensitivity of the biosensor used ^[26][57]. In this regard, it is clear that non-enzymatic sensors that provide direct cholesterol signals have some advantages compared to biosensors, such as more advanced methodological features, simpler structure, lower cost, and longer shelf life.

2. Cholesterol Determination Methods in Foods

Recently, many new methods have been developed to detect the amount of cholesterol in various materials. Cholesterol can be detected by a variety of analytical methods, such as gravimetry, colorimetry, fluorimetry, chromatography, and enzymatic and non-enzymatic sensors. These methods can be examined in four main categories: (1) classic chemical tests, (2) colorimetric and fluorometric enzymatic tests frequently used in test kits and automatic plate readers, (3) analytical methods such as gas and liquid chromatography or mass spectrometry, and (4) enzymatic and non-enzymatic sensors. Classical chemical methods are relatively simple and inexpensive to apply compared to others but require multi-step procedures. Enzymatic assays involve the use of expensive enzymes, but their limits of detection (LODs) are generally low. Methods such as chromatography and mass spectrometry are the most accurate and sensitive methods. However, they require expensive equipment and long sample preparation preprocessing ^[27][58].

2.1. Detection of Cholesterol Using Enzymatic and Non-Enzymatic Methods

A wide variety of methods have been reported to monitor cholesterol in biological fluids and foods ^{[28][29][30][31]}[59,60,61,62]. Enzymatic and non-enzymatic electrochemical determination methods to determine cholesterol have also been recently introduced ^[32][24]. Cholesterol was thought to be electrochemically inactive until the late 1980s ^[33][63]. In 2005, methods that first provided the direct oxidation of cholesterol and then indirect cholesterol oxidation methods (using an electron mediator) were developed ^[34][35][64,65]. Cholesterol oxidase, less commonly cholesterol esterase, and cholesterol modified with nanomaterials have been widely evaluated as sensing materials in electrochemical sensors ^[26][57]. Each of these approaches varies significantly, and their general use is quite limited because cholesterol oxidation products must be identified separately for each chosen electrolysis environment. Many new methods have been introduced for the characterization of direct oxidation products of cholesterol in non-aqueous media ^[33][35][36][63,65,66].

2.2. Determination of Cholesterol in Foods by Enzymatic Methods

This type of analysis relies on the presence of an enzymatic reaction to determine total cholesterol levels in food or other materials. The first enzymatic test capable of detecting cholesterol in serum was introduced in 1974 ^[37][67]. Enzymatic tests have been widely used since then, and more recently test kits and automated analyzers are now widely used ^[38][68]. In these methods, esterified cholesterol is first hydrolyzed to free cholesterol by cholesterol esterase. The resulting free cholesterol is then oxidized to cholesta-4-en-3-one by the cholesterol oxidase enzyme. Hydrogen peroxide is produced as a byproduct in this reaction and can be easily detected using high-sensitivity colorimetric or fluorometric probes ^[37][39][40][67,69,70].

2.3. Detection of Cholesterol in Foods by Chromatographic Methods

2.3.1. Detection of Cholesterol in Foods with HPLC

The most common techniques for the nonenzymatic removal of cholesterol from foods are HPLC and other techniques such as GC-MS and LC-MS ^[6][41][38,71]. These mentioned methods are extremely sensitive and selective; however, they require complex sample preparation procedures and expensive equipment. The measurement of total cholesterol is generally made using chromatographic or enzymatic methods. The most commonly used method is gas chromatography, although some HPLC methods are also popular ^[42][43][72,73]. However, the determination of cholesterol by HPLC has received less attention than measurement by GC. The application of HPLC with UV detection to determine cholesterol has been limited in a complex sample environment, as the poor absorption of cholesterol at low wavelengths poses a problem in spectrophotometric detection ^[44][74]. It has been determined that gas chromatography (GC) is more sensitive, especially in determining cholesterol in food matrices ^{[45][46][47][48][49][50]}[75,76,77,78,79,80]. These drawbacks encountered in HPLC measurements can be easily overcome by using high-performance liquid chromatography (HPLC) and especially reversed-phase HPLC. Compared to HPLC, cholesterol determination by GC is more laborious. For example, it has disadvantages such as the derivatization of cholesterol compounds, the need to verify the reliability of measurement using internal standards before use, sample preparation being time-consuming (involving steps such as saponification and extraction, with a chromatographic run taking approximately 25 min), and being a costly method. In addition, the GC instrument is operated at a higher temperature than HPLC, which induces the formation of cholesterol oxides ^[6][51][38,81]. UHPLC and HPLC may be ideal analytical techniques for measuring cholesterol in food matrices because they are more sensitive, cost-effective, and less time-consuming than other traditional methods. The most common detector used for cholesterol detection in HPLC is the diode array detector (DAD). Studies have found that the most suitable HPLC detector to measure cholesterol levels in foods is a diode array detector (DAD), and other detectors such as ultraviolet (UV), fluorescence detection, evaporative light-scattering detection, infrared detection, and electrochemical detection have also been reported to be suitable for measurement ^{[6][45][46][47][50][52][53]}[38,75,76,77,80,82,83]. The most convenient and often used method for sample preparation in liquid chromatography is the direct saponification of the sample followed by extraction of the unsaponifiable residue into a nonpolar solvent ^[54][86]. In this method, direct saponification is preferred because of the possibility of converting non-polar fatty acid esters to polar products by effectively removing them by multiple extractions with n-hexane ^[6][54][55][38,86,87]. Saponification and extraction processes are very important in determining the cholesterol ratio by HPLC. The saponification process is one of the most important steps in obtaining cholesterol purified from other components. In this process, potassium hydroxide is the most common solvent used to separate cholesterol from fatty acids ^[45][52][56][75,82,88]. The mixture is then washed with ultrapure water to remove these compounds, and the resulting cholesterol remains in the extracted solution layer for analysis ^[45][75].

2.3.2. Detection of Cholesterol in Foods with GC-MS

Gas chromatography (GC) is also one of the most commonly used analytical techniques for the quantification of cholesterol and other sterols. However, although GC columns are a very effective method for the separation of cholesterol, sometimes problems may arise in the separation of cholesterol due to its similarities with other sterols ^[52][56][82,88]. It is widely accepted that this method is more reliable, sensitive, and accurate than other methods. In chromatographic columns, additional selectivity can be added to separate cholesterol from inhibitory sterols. Additionally, another advantage of this method is that a low volume of sample (tens of mL) is required for analysis ^[57][89]. The AOAC has published 994.10, an official method for the analysis of cholesterol in foods by GC-flame ionization detector after saponification and derivatization with trimethylchlorosilane ^[58][90]. In general, procedures applied in GC require the extraction of total lipids, separation of solvents, saponification of cholesterol esters, detailed solvent extraction of unsaponifiable material, repeated washing concentration of the analyte, and appropriate derivatization before GC analysis ^[58][90]. These process steps are quite burdensome in terms of both labor and materials and require increasingly expensive supply, recovery, and disposal costs. Although newer methods based on direct saponification of the sample have been developed and some steps have been eliminated, this method remains laborious and costly ^[59][91].

2.3.3. Electrospray Ionization Tandem Mass Spectrometer (ESI)

ESI is not an effective method for ionization to measure neutral free sterol molecules such as cholesterol. The molecular ions of the sterol molecules are easily fragmented in the [M + H]⁺ ion source. For that reason, they are very difficult to detect in the matrix using this method. In contrast, cholesterol ester species tend to form more stable ammonium adduct ions [M + NH₄]⁺, which can be detected successfully ^[60][92].

2.3.4. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI)

MALDI is an effective ionization method for neutral free sterol molecules. Recent studies have provided increasing evidence that MALDI-MS can be used for cholesterol measurements. In a study conducted by Hidaka et al. ^[61][93], MALDI-TOF (time-of-flight) MS was used to analyze cholesterol in human serum lipoproteins.

2.3.5. Ambient Ionization Mass Spectrometer

In recent years, a method known as ambient ionization mass spectrometry (AIMS) has been developed for the chemical analysis of cholesterol in biological systems ^[62][94]. The ambient ionization technique operates at atmospheric pressure, in real time, and requires minimal sample pretreatment for rapid mass spectrometric analysis. Desorption electrospray ionization (DESI) can be used directly for real-time direct analysis (DART) and quantitative cholesterol measurements. It is also a technology that is twice as fast and more effective than direct ESI approaches for cholesterol ionization ^[63][95].

2.3.6. Removal of Cholesterol from Foods by Nonenzymatic Methods

Cholesterol replacement therapy is required for the treatment of some inborn errors of metabolism. Therefore, most cholesterol determination techniques target blood and blood serum. The determination of cholesterol in foods is extremely important for health. Over the last decade, a wide variety of cholesterol sensors have been developed, including enzymatic, non-enzymatic, and redox mediator-based sensors. In enzymatic systems, the hydrolysis of cholesterol ester is catalyzed by using enzymes such as cholesterol oxidase (ChOx) or cholesterol esterase (CE), resulting in the formation of fatty acids and free cholesterol. Enzymatic sensors demonstrate high sensitivity and selectivity. However, the enzymes have a short lifespan and are easily denatured during immobilization. Additionally, their activities are easily affected by temperature, pH value, and toxic chemicals ^[64][65][96,97]. Non-enzymatic cholesterol sensors do not face the same limitations and problems as enzymatic ones. Electrode surfaces modified with metals, metal oxides, or composites have electrocatalytic functions. The most important feature of non-enzymatic electrodes is the use of nanomaterials with high surface-to-volume ratios that provide good interaction with external reagents, high conductivity, and excellent biocompatibility.

2.4. Electrochemical Sensors

Electrochemical sensors are preferred because they are simple, are low-cost, have high accuracy, and have high and fast sensitivity levels. Carbon-based electrodes are widely used in electrochemical analysis because of their advantages such as the ease of surface modification and low background current ^[66][67][106,107]. The electron transfer rates of electrochemical sensors are generally lower than electrodes made of noble metals ^[68][108]. The electrochemical activity of carbon-based electrodes against some analytes can be increased by the anodic oxidation of the surfaces, resulting in the formation of new oxidized functional groups. In bare electrodes, a high voltage is required for cholesterol oxidation ^[33][63]. Overvoltage occurring in chemically modified electrodes (CMEs), popular in electro-analytical chemistry, can be significantly reduced using electrochemical sensors.

2.5. Possibilities of Using Biosensors in Measuring Cholesterol in Foods

Among the various existing methods used to detect cholesterol, biosensors are a relatively simpler, faster, more sensitive, and more specific method ^[30][69][61,112]. Biosensors are analytical devices that consist of a transducer and a biological element. Bioelements such as enzymes, antibodies, nucleic acids, receptors, organelles, and microorganisms interact with the analyte under study, and the concentration of substances or other biological response parameters are converted into an electrical signal ^[70][113]. In biosensor production, the immobilization of enzymes on electrodes is important. The high performance of the amperometric biosensor simply immobilizes the enzyme on the electrode and stimulates electron transfer in sensor fabrication using mediators, promoters, or other special materials. The majority of cholesterol biosensors have been developed based on the electrochemical reduction of hydrogen peroxide (H₂O₂) due to their simplicity and specificity ^[71][12]. In a study conducted by Ferri et al. ^[72][116], the behavior of a multi-enzymatic electrodic system for the detection of glucose and choline based on horseradish peroxidase (HRP) was investigated. The electrodic system captured by horseradish peroxidase quickly detected the presence of these analytes, which are involved in the reactions in which hydrogen peroxide is produced, even without the need for additional mediators ^[73][117].