Immobilization Of Cholesterol Oxidase Research Articles

Development of an electrochemical biosensor for non-invasive cholesterol monitoring via microneedle-based interstitial fluid extraction

In this study, we present the development of an innovative electrochemical biosensor integrated into a microneedle-based system for non-invasive and sensitive quantification of cholesterol levels in interstitial fluid (ISF). The biosensor employs a graphene-based electrode with a polyelectrolyte interlayer to immobilize cholesterol oxidase (ChOx), enabling selective cholesterol detection. Graphene oxide is electrochemically reduced to form a conductive layer, and PANI is chosen as the optimal polyelectrolyte for ChOx immobilization. The biosensor's performance is thoroughly evaluated, demonstrating excellent sensitivity, stability, and selectivity. Furthermore, the biosensor is successfully applied to skin-mimicking agarose gel and porcine skin, showcasing its potential for real-world interstitial fluid extraction and cholesterol monitoring. The integrated microneedle-based system offers a promising approach for non-invasive monitoring of cholesterol levels, with implications for personalized healthcare diagnostics.

A Sensitive Enzymatic Electrochemical Biosensor for Cholesterol Based on Cobalt Ferrite@Molybdenum Disulfide/Gold Nanoparticles.

Cholesterol is essential in biological systems, and the level of cholesterol in the body of a person acts as a diagnostic marker for a variety of diseases. So, in this work, we fabricated an enzymatic electrochemical biosensor for cholesterol using cobalt ferrite@molybdenum disulfide/gold nanoparticles (CoFe2O4@MoS2/Au). The synthesized composite was used for the determination of cholesterol by voltametric methods. The electroactive material CoFe2O4@MoS2/Au was successfully verified from the physiochemical studies such as XRD, Raman, FT-IR, and XPS spectroscopy along with morphological FESEM and HRTEM characterization. CoFe2O4@MoS2/Au showed outstanding dispersion in the aqueous phase, a large effective area, good biological compatibility, and superior electronic conductivity. The microflower-like CoFe2O4@MoS2/Au was confirmed by scanning electron microscopy. The image of transmission electron microscopy showed decoration of gold nanoparticles on CoFe2O4@MoS2 surfaces. Furthermore, a one-step dip-coating technique was used to build the biosensor used for cholesterol detection. In addition to acting as an enabling matrix to immobilize cholesterol oxidase (ChOx), CoFe2O4@MoS2/Au contributes to an increase in electrical conductivity. The differential pulse voltammetry method was used for the quantitative measurement of cholesterol. The calibration curve for cholesterol was linear in the concentration range of 5 to 100 μM, with a low limit of detection of 0.09 μM and sensitivity of 0.194 μA μM-1 cm-2. Furthermore, the biosensor demonstrates good practicability, as it was also employed for identifying cholesterol in real samples with acceptable selectivity and stability.

Relay run property of immobilized cholesterol oxidase on magnetic biochar for sterol removal

This study investigated the biocatalytic performance of immobilized cholesterol oxidase (CHOD) on magnetite-based carbon (MBC) for degrading cholesterol. The results showed that MBC-CHOD exhibited higher activity and good affinity towards substrate compared to free enzyme and other immobilized enzymes. Mass spectra analysis revealed that MBC-CHOD damaged the main structure of cholesterol, benefitting the further biological treatment. The study proposes a Fenton process mechanism by which H2O2 is transferred to free radicals such as ·OH under acidic conditions, promoting further substrate degradation. This suggests that MBC-CHOD has a relay run property leading to high degradation of cholesterol. Molecular docking indicates that cholesterol preferentially binds to TYR-28 residue and LYS-138 residue in CHOD through hydrogen bonds. Overall, MBC-CHOD proved to be a promising candidate for efficient and sustainable cholesterol degradation.

O-Fenilendiamin ve Benzokinon Bazlı Polimer Filmle Oluşturulan Kolesterol Biyosensörünün Elektrokimyasal Karakterizasyonu

Aim: The detection of cholesterol concentration in the blood is an important parameter in the diagnosis and follow-up of many diseases, including cardiovascular and hepatobiliary diseases. Nowadays, various methods have been used for the routine analysis of cholesterol, including spectrophotometric, high-performance liquid chromatography (HPLC), and amperometric methods. Since biosensors have advantages such as high sensitivity, fast response, low cost, small size, continuous online detection, and reproducible results, it is aimed to develop a biosensor method for cholesterol determination. Methods: Polymer film immobilization was performed on the gold electrode surface electrochemically in an acetonitrile-water medium containing o-phenylenediamine and benzoquinone. Cholesterol oxidase (ChOx, E.C. 1.1.3.6) enzyme was immobilized on the polymer film-immobilized electrode surface by the self-forming monolayer method. Optimization and characterization studies were carried out for the determination of cholesterol with the prepared electrodes. Results: The determination of cholesterol was performed via monitoring the oxidation current of enzymatically produced H2O2 at 0.70 V Ag/AgCl. Optimum biosensor conditions were determined for phosphate buffer at 0.1M, pH 7.0, and 30°C for the Au/oPD-BQ/ChOx enzyme electrode. The linear working range is 9.8×10-6-1.1×10-2 mM, and the cholesterol detection limit is 9.8×10-6 mM. The analysis of total cholesterol in solutions was performed using the proposed Au/oPD-BQ/ChOx electrode. Conclusion: A convenient, simple, reproducible, and stable method was developed for the determination of cholesterol by immobilizing cholesterol oxidase on the prepared electrode surface through a polymer film. The sensor we designed can be expanded to improve the detection of many analytes in the clinic.

Direct Electrochemistry of Cholesterol Oxidase Immobilized on PEDOT Functionalized Screen-Printed Electrodes

To address the problems of high cost and cumbersome preparation process in the current construction of cholesterol biosensors, a third-generation electrochemical cholesterol biosensor with a facile preparation method, low cost, enhanced sensitivity, and wide linear range was investigated in this paper. The cholesterol biosensor was constructed by immobilizing cholesterol oxidase (ChOx) on a poly-3,4-ethylenedioxythiophene (PEDOT) modified screen-printed electrode (SPE). This study showed a very simple fabrication process. The electrodes were constructed only in three steps, including in situ electropolymerization of PEDOT, enzyme introduction and encapsulation of the nafion membrane. The successful modification of PEDOT was demonstrated by Energy Dispersive X-ray spectroscopy (EDX) and Fourier-Transform Infrared spectroscopy (FT-IR). The morphological characterization and electrochemical analysis showed that the presence of PEDOT not only provided more anchoring sites for immobilizing enzymes, but also its high electrocatalytic activity enabled direct electron transfer (DET) between the ChOx and the electrode surface. The linear range for the actual detection of cholesterol was 50–800 μM, the sensitivity was 1.34 μA mM−1, and the electrical signal was not affected by interfering substances such as uric acid, glucose, dopamine, and ascorbic acid. Therefore, the cholesterol biosensor constructed in this paper was expected to achieve large-scale applications.

Cholesterol oxidase-immobilized MXene/sodium alginate/silica@n-docosane hierarchical microcapsules for ultrasensitive electrochemical biosensing detection of cholesterol.

Electrochemical biosensors usually suffer from the deterioration of detection sensitivity and determination accuracy in a high-temperature environment due to protein denaturation and inactivation of their biological recognition elements such as enzymes. Focusing on an effective solution to this crucial issue, we have developed cholesterol oxidase-immobilized MXene/sodium alginate/silica@n-docosane hierarchical microcapsules as a thermoregulatory electrode material for electrochemical biosensors to meet the requirement of ultrasensitive detection of cholesterol at high temperature. The microcapsules were first fabricated by microencapsulating n-docosane as a phase change material (PCM) in a silica shell, followed by depositing a biocompatible sodium alginate layer, wrapping with electroactive MXene nanosheets and then immobilizing cholesterol oxidase as a biological recognition element for electrochemical biosensing. The fabricated composites not only exhibited a layer-by-layer hierarchical microstructure with the desired chemical and biological components, but also obtained a high latent-heat capacity of over 133 J g-1 for thermal management through reversible phase transitions of its PCM core. A bare glassy carbon electrode was modified with the developed composites to serve for the cholesterol biosensor. This enables the modified electrode to obtain an in situ thermoregulatory ability to regulate the microenvironmental temperature surrounding the electrode, effectively preventing the protein denaturation of cholesterol oxidase and minimizing heat impact on biosensing performance. Compared to conventional cholesterol biosensors without a PCM, the developed biosensor achieved a higher sensitivity of 4.63 μA μM-1 cm-2 and a lower limit of detection of 0.081 μM at high temperature, providing highly accurate and reliable detection of cholesterol for real biological samples over a wide temperature range.

Highly sensitive cholesterol biosensor based on electron mediator thionine and cubic-shaped Cu2O nanomaterials

The preparation of an electrochemical biosensor applicable for the highly sensitive detection of cholesterol content in biological samples in clinical practice is studied in this paper. Commonly used in biosensors, nano-cuprous oxide (Cu2O) contains active electron-hole pairs and a large specific surface area, while thionine (TH) improves the efficiency of electron transfer between the enzyme and the electrode. In this study, a cholesterol biosensor, cholesterol oxidase (ChOx)/thionine/nano-cuprous oxide/ glassy carbon electrode (GCE) (ChOx/TH/Cu2O/GCE), is prepared using cubic-shaped Cu2O as the nanomaterial, TH as an electron mediator, and chitosan (CS) as a cross-linking agent to immobilize cholesterol oxidase. The results show that Cu2O nanoparticles (NPs) are successfully synthesized, and the cholesterol biosensor prepared from synthesized Cu2O NPs exhibits excellent electrochemical characteristics, good linearity (R2 = 0.997) over a substrate concentration range of 10 to 1000 µM, a sensitivity of 70.22 µA µM−1 cm−2, and a low detection limit of 0.0018 µM for the substrate. The ChOx, which immobilizes on the composite electrode, shows a strong affinity for cholesterol due to its low Michaelis–Menten constant (Km) value (25.359 µM). Moreover, with strong anti-interference, good reproducibility, and stability, the cholesterol biosensor can achieve a recovery rate of 99.71 % to 107.75 % according to a recovery test of actual spiked samples. Thus, the prepared nanomaterial-based cholesterol biosensor can be applied to the highly sensitive detection of cholesterol in actual biological samples.

Improvement on Reusability, Storage Stability and Thermal Stability of Magnetic Graphene Oxide- Immobilized Cholesterol Oxidase

Improvement on Reusability, Storage Stability and Thermal Stability of Magnetic Graphene Oxide- Immobilized Cholesterol Oxidase

An amperometric cholesterol biosensor based on immobilization of cholesterol oxidase onto titanium dioxide nanoparticles

Cholesterol is an important amphipathic molecule and essential structural constituent of all animal cells. It is made up of a hydrocarbon chain, four hydrocarbon ring structure and a hydroxyl group. It is also used in Vitamin D biosynthesis. Cholesterol is of two types: LDL (low-density lipoproteins, bad cholesterol) and HDL (high-density lipoproteins, good cholesterol. High cholesterol increase risk of coronary heart disease. Proposed work describes a unique approach of immobilizing cholesterol oxidase onto TiO2NPs, modified Pencil Graphite electrode and its application in construction of an amperometric biosensor for determination of serum cholesterol.

Immobilization of Cholesterol Oxidase in Chitosan Magnetite Material for Biosensor Application

Immobilization of Cholesterol Oxidase in Chitosan Magnetite Material for Biosensor Application

Immobilization of Cholesterol Oxidase from Streptomyces Sp. on Magnetite Silicon Dioxide by Crosslinking Method for Cholesterol Oxidation.

Enzymatic biosensor has been paid much attention to the research fields due to its advantage in medical application. As one of the application, we determined the optimum value of cholesterol oxidase against cholesterol. In this work, we studied the behavior of cholesterol oxidation by enzymatic reaction to get the optimum condition for cholesterol oxidation. The enzyme that used were in two form, free cholesterol oxidase, and immobilized cholesterol oxidase. Cholesterol oxidase was produced from Streptomyces sp. by using solid state fermentation method and identified had high enzyme activity to be 5.12U/mL. Cholesterol oxidase was simultaneously crosslinked immobilized onto magnetite coated by silicon dioxide (M-SiO2). The support was characterized by Fourier transform infrared (FTIR) to determine the functional group of modified particle and scanning electron microscope (SEM) to observe the morphological or our prepared particle. Cholesterol oxidase sensitivity to substrate was analyzed by using HPLC with different interval time measurements. The oxidation of cholesterol by free enzyme and immobilized enzyme was also investigated. The best sensitivity of cholesterol oxidase was estimated to oxidize Cso (concentration of substrate) 1.46mM of substrate with Ce (concentration of enzyme) 20mg/mL for 180min. Final oxidation value of cholesterol by immobilized enzyme was greater than 60%. The results of this study revealed that immobilized enzyme for cholesterol oxidation was stable, reproducible, and sensitive.

Layer-by-Layer Films of Gold Nanoparticles and Carbon Nanotubes for Improved Amperometric Detection of Cholesterol.

Distinct architectures of layer-by-layer (LbL) films made of carbon nanotubes and gold nanoparticles were investigated to serve as the matrix to immobilize cholesterol oxidase, with which cholesterol could be detected using amperometry. The gold nanoparticles were synthesized by metal reduction stabilized in poly(allylamine hydrochloride) (PAH) providing a stable AuNPs-PAH suspension, while multi-walled carbon nanotubes (CNTs) were functionalized with carboxylic groups to obtain an aqueous suspension. The LbL films were deposited on ITO, with a cushion film of PAH and poly(vinyl sulfonic acid) (PVS). Owing to the synergy between CNTs and AuNPs, the electrode ITO/(PAH/PVS)₂ (AuNPs-PAH/CNTs)10 was selected for immobilization of cholesterol oxidase (ChOx). This sensor could detect cholesterol with a limit of detection of 14.8 μmol L-1 and sensitivity of 36.47 (μA cm-2)·(mmol L-1)-1. It was also able to determine cholesterol in egg yolk with a recovery of 97.7%.

Colorimetric point-of-care detection of cholesterol using chitosan nanofibers

Recent developments in biosensor-related research have provided economic and highly sensitive biosensors for numerous biological analytes. Several strategies have evolved for developing a sensitive biosensor for cholesterol detection. In this study, we have developed a point-of-care, chitosan nanofiber-based cholesterol biosensor, involving colorimetric detection of the analyte. Chitosan nanofibers, fabricated using electrospinning, were utilized for immobilizing cholesterol oxidase and peroxidase enzymes. A uniform and bead-free chitosan nanofibers were obtained using chitosan and polyvinyl alcohol (Cs: PVA), at the ratio of 0.7:1 w/w, in a solvent system containing 10%v/v of methanol in 63% acetic acid. The nanofibers were characterized using scanning electron microscopy (SEM), which revealed the formation of smooth fibers, with an average diameter of 60–90 nm. The fabricated nanofibers offered a greater surface area for immobilizing high quantities of enzymes and demonstrated the potential to be developed into a strip-based intervention, based on a simple, visual detection system. A colorimetric detection method was developed using the chromogenic substrate, 3,3́,5,5́-tetramethylbenzidine hydrochloride. Analysis by UV–vis spectrophotometry demonstrated a linear increase in the absorbance, with increasing concentrations of cholesterol. The intensity of color change, as a function of cholesterol concentration was used for developing a color gradient scale for the investigated biosensor.

A Fiber Optic Biosensor Based on Hydrogel-Immobilized Enzyme Complex for Continuous Determination of Cholesterol and Glucose.

A multiparameter fiber optic biosensor for continuous determination of cholesterol and glucose was developed. This sensor was based on poly(N-isopropylacrylamide) (PNIPAAm)-immobilized glucose oxidase (GOx) complex (PIGC) and immobilized cholesterol oxidase (COD). The immobilized COD catalysis to the oxidation of cholesterol and PIGC catalysis to the oxidation of glucose could be performed at different temperatures. Therefore, the sensor could detect cholesterol and glucose continuously by changing temperature. The optimal detection conditions for glucose were achieved with pH6.5, 30°C, and 10mg GOx (in 100-mg carrier), and those for cholesterol were achieved with pH7.5, 33°C, and 25mg COD (in 250-mg carrier). The sensor has the cholesterol detection range of 20-250mg/dL and the glucose detection range of 50-700mg/dL. This biosensor has outstanding repeatability and selectivity, and the detection results of the practical samples are satisfactory.

Immobilization of Cholesterol Oxidase: An Overview

Background:Cholesterol oxidases are bacterial oxidases widely used commercially for their application in the detection of cholesterol in blood serum, clinical or food samples. Additionally, these enzymes find potential applications as an insecticide, synthesis of anti-fungal antibiotics and a biocatalyst to transform a number of sterol and non-sterol compounds. However, the soluble form of cholesterol oxidases are found to be less stable when applied at higher temperatures, broader pH range, and incur higher costs. These disadvantages can be overcome by immobilization on carrier matrices.Methods:This review focuses on the immobilization of cholesterol oxidases on various macro/micro matrices as well as nanoparticles and their potential applications. Selection of appropriate support matrix in enzyme immobilization is of extreme importance. Recently, nanomaterials have been used as a matrix for immobilization of enzyme due to their large surface area and small size. The bio-compatible length scales and surface chemistry of nanoparticles provide reusability, stability and enhanced performance characteristics for the enzyme-nanoconjugates.Conclusion:In this review, immobilization of cholesterol oxidase on nanomaterials and other matrices are discussed. Immobilization on nanomatrices has been observed to increase the stability and activity of enzymes. This enhances the applicability of cholesterol oxidases for various industrial and clinical applications such as in biosensors.

Immobilization of cholesterol oxidase: An overview

Immobilization of cholesterol oxidase: An overview

LSPR- and SPR-Based Fiber-Optic Cholesterol Sensor Using Immobilization of Cholesterol Oxidase Over Silver Nanoparticles Coated Graphene Oxide Nanosheets

Fabrication and characterization of a highly sensitive fiber-optic cholesterol sensor utilizing cholesterol oxidase (ChOx) have been carried out. The operation of the sensor is based on combined phenomenon of localized and propagating surface plasmons. Two different immobilization schemes of enzyme ChOx have been used. In one, the enzyme is entrapped in the hydrogel and in second the enzyme is immobilized over the graphene oxide (GO) nanosheets. The synthesis of GO is confirmed by XRD, TEM, and Raman spectroscopy and the binding between ChOx and GO is verified through the Raman spectroscopy and Fourier transform infrared spectroscopy techniques. Following three types of probes having different configurations are fabricated and compared: 1) enzyme entrapped gel layer over silver coated unclad fiber; 2) enzyme immobilized over silver and GO coated unclad fiber; and 3) enzyme immobilized over silver, GO, and silver nanoparticles coated unclad fiber. The performance of all these probes have been compared in terms of operating range, sensitivity and limit of detection. The sensors have many advantages, such as compact size, modest cost, high sensitivity, high selectivity, and capability of remote sensing.

Graphene and Au NPs co-mediated enzymatic silver deposition for the ultrasensitive electrochemical detection of cholesterol

Cholesterol is an essential ingredient in mammals, and serum cholesterol is a major component of atherosclerotic plaques. The level of cholesterol in human serum has become an important index for clinical diagnosis and prevention of cardiovascular disease. In this paper, a simple and ultrasensitive cholesterol biosensor based on graphene oxide (GO) and gold nanoparticles (Au NPs) co-mediated enzymatic silver deposition was designed by immobilizing cholesterol oxidase (CHOD), cholesterol esterase (CHER) and GO onto the surface of Au NPs modified screen-printed carbon electrode (SPE). Under the synergistic effect of CHER, CHOD and GO, the cholesterol was hydrolyzed to generate hydrogen peroxide, which can reduce the silver (Ag) ions in the solution to metallic Ag which deposited on the surface of Au NPs modified SPE. The ultrasensitive detection of cholesterol was achieved by anodic stripping voltammetry measurement of the enzymatically deposited Ag. Under optimal conditions, the anodic stripping peak current of Ag increased with the increasing cholesterol concentration in the range from 0.01μg/mL to 5000μg/mL with a limit of detection of 0.001μg/mL (S/N = 3). In addition, the ultrasensitive cholesterol biosensor exhibited higher specificity, acceptable reproducibility and excellent recoveries for cholesterol detection.

A temperature-triggered fiber optic biosensor based on hydrogel-magnetic immobilized enzyme complex for sequential determination of cholesterol and glucose

This work described a temperature-triggered fiber optic biosensor for sequential detection of cholesterol and glucose based on poly(N-isopropylacrylamide)-co-acrylamide)(P(NIPAAm-co-AAm))-magnetic immobilized glucose oxidase(GOD) complex (PMIGC) and magnetic immobilized cholesterol oxidase (COD). At 38°C, the sensor will selectively detect cholesterol concentration with the detection range of 25–250mg/dL since P(NIPAAm-co-AAm) is known to shrink above its lower critical solution temperature (LCST) of 36°C, and will separate GOD from analytes so that PMIGC has no catalysis effect on glucose. When the temperature was switched to 25°C (below LCST), PMIGC could catalyze the oxidation of glucose since GOD will be exposed to analytes due to the swell of P(NIPAAm-co-AAm). This sensor can be used for the detection of glucose with the detection range of 50–700mg/dL. The optimal detection conditions for cholesterol were achieved with pH 7.0, 40°C and 10mg COD (in 75mg carrier), and those for glucose were achieved with pH6.5, 35°C and 12mg GOD (in 90mg carrier). The biosensor proposed is shown to have outstanding repeatability, selectivity and yield satisfactory detection results on practical samples.

In situ synthesis of cylindrical spongy polypyrrole doped protonated graphitic carbon nitride for cholesterol sensing application

Herein, we demonstrate the exfoliation of bulk graphitic carbon nitrides (g-C3N4) into ultra-thin (~3.4nm) two-dimensional (2D) nanosheets and their functionalization with proton (g-C3N4H+). The layered semiconductor g-C3N4H+ nanosheets were doped with cylindrical spongy shaped polypyrrole (CSPPy-g-C3N4H+) using chemical polymerization method. The as-prepared nanohybrid composite was utilized to fabricate cholesterol biosensors after immobilization of cholesterol oxidase (ChOx) at physiological pH. Large specific surface area and positive charge nature of CSPPy-g-C3N4H+ composite has tendency to generate strong electrostatic attraction with negatively charged ChOx, and as a result they formed stable bionanohybrid composite with high enzyme loading. A detailed electrochemical characterization of as-fabricated biosensor electrode (ChOx-CSPPy-g-C3N4H+/GCE) exhibited high-sensitivity (645.7 µAmM−1 cm−2) in wide-linear range of 0.02–5.0mM, low detection limit (8.0μM), fast response time (~3s), long-term stability, and good selectivity during cholesterol detection. To the best of our knowledge, this novel nanocomposite was utilized for the first time for cholesterol biosensor fabrication that resulted in high sensing performance. Hence, this approach opens a new prospective to utilize CSPPy-g-C3N4H+ composite as cost-effective, biocompatible, eco-friendly, and superior electrocatalytic as well as electroconductive having great application potentials that could pave the ways to explore many other new sensors fabrication and biomedical applications.