Cholesterol In Human Serum Samples Research Articles

Fe-Ni dual-single atoms nanozyme with high peroxidase-like activity for sensitive colorimetric and fluorometric dual-mode detection of cholesterol

Cholesterol is widely existed in nerve myelin sheath and various membrane structures, whose abnormal level would deteriorate human cells or even cause diseases. Herein, Fe-Ni dual-single-atom nanozyme was efficiently incorporated into N-doped carbon nanosheets (FeNi DSAs/N-CSs) by a simple calcination method. Its nanozyme activity and catalytic mechanism were investigated in details. The FeNi DSAs/N-CSs nanozyme showed superior peroxidase-like property, which was applied for the dual-mode determination of hydrogen peroxide (H2O2) and cholesterol. The colorimetric/fluorometric assays of H2O2 displayed the linear ranges of 1–50 mM and 5–40 mM with low limits of detection of 0.45 mM and 3.33 mM, respectively. In parallel, there exhibited the linear ranges of 0.5–5.0 mM and 0.25–5.0 mM for the colorimetric/fluorometric analysis of cholesterol, coupled with the limits of detection down to 0.19 mM and 0.044 mM, respectively. This work provided a rapid, cost-effectiveness and simple colorimetric/fluorometric method for sensitive dual-mode detection of cholesterol in human serum samples.

Enzymatic Reactions in a Lab-on-Valve System: Cholesterol Evaluations.

The micro sequential injection analysis / lab-on-valve (µSIA-LOV) system is a miniaturized SIA system resulting from the implementation of a lab-on-valve (LOV) atop of the selection valve. It integrates the detection cell and the sample processing channels into the same device, promoting the reduction of reagent consumption and waste generation, the improvement of the versatility, and the reduction of the time of analysis. All of these characteristics are really relevant to the implementation of enzymatic reactions. Additionally, the evaluation of cholesterol in serum samples is widely relevant in clinical diagnosis, since higher values of cholesterol in human blood are actually an important risk factor for cardiovascular problems. An automatic methodology was developed based on the µSIA-LOV system in order to evaluate its advantages in the implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase and peroxidase. Considering these reactions, the developed methodology was also used for the evaluation of cholesterol in human serum samples, showing reliable and accurate results. The developed methodology presented detection and quantification limits of 1.36 and 4.53 mg dL−1 and a linear range up to 40 mg dL−1. This work confirmed that this µSIA-LOV system is a simple, rapid, versatile, and robust analytical tool for the automatic implementation of enzymatic reactions performed by cholesterol esterase, cholesterol oxidase, and peroxidase. It is also a useful alternative methodology for the routine determinations of cholesterol in real samples, even when compared with other automatic methodologies.

Luminol and gold nanoparticle-co-precipitated reduced graphene oxide hybrids with long-persistent chemiluminescence for cholesterol detection.

Herein, dual-functionalized reduced graphene oxide (rGO) hybrids (rGO/AuNP/luminol) with gold nanoparticles (AuNPs) and luminol were prepared viaπ-π conjugation and electrostatic interaction. The as-prepared rGO/AuNP/luminol hybrids exhibited enhanced and long-persistent chemiluminescence (CL) performance without decay for one hour when mixed with H2O2; this was due to the generation of ˙OH, O2˙-, 1O2, and π-C[double bond, length as m-dash]C˙ species. A sensitive and selective CL sensor based on the rGO/AuNP/luminol hybrid CL enhancing system was developed for cholesterol quantitation in the linear range from 0.71 to 11.43 μM with a detection limit of 0.55 μM. Furthermore, the proposed method was successfully exploited for the determination of cholesterol in human serum samples.

Spectrophotometric Determination of Cholesterol in Human Serum Samples

A sensitive spectrophotometric method for the determination of cholesterol in serum samples was described. 4-Hydroxybenzaldehyde was used for spectrophotometric determination of cholesterol. The method based on the formation of coloured compound showed maximum absorbance at 525 nm. Beer ’ s law was obeyed in the concentration range 1.0 – 30.0 µg/ml with detection limit 0.14 µg/ml. The method was applied for the analyses of cholesterol in human serum samples. The results were compared with biolabo kit and biolis automatic instrument as a standard method which shows good agreement between them.

Cholesterol aided etching of tomatine gold nanoparticles: A non-enzymatic blood cholesterol monitor

Colloidal gold is extensively used for molecular sensing because of the wide flexibilities it offers in terms of modifications of the gold nanoparticles (GNPs) surface with a variety of functional groups. We describe a simple, enzyme free assay for the detection of cholesterol, and demonstrate its applicability by estimating cholesterol in human serum samples. To enable cholesterol detection, we functionalized GNPs with tomatine, a glycoalkaloid found in the leaves and stem of tomato plants. The binding of cholesterol onto tomatine functionalized gold nanoparticles (TGNPs) was characterized by a blue shift in the plasmon absorption spectra (SPR) followed by reduction in the particle size. The TGNPs have been core etched with increasing concentration of cholesterol and with 800ng/mL of cholesterol particles in the size range of 10–12nm have been obtained. This behavior was attributed to the enhanced hydrophobicity of the surface acquired by cholesterol binding resulting in the folding or shrinkage of molecule in turn leading to core etching. The method was successfully applied for the detection of cholesterol in real samples and agrees well with values obtained from the conventional method. Because of its significant plasmonic shift and simplicity, this biosensor could be used for cholesterol detection as it does not demand either any hazardous and costly chemicals or any complex synthetic routes.

Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode

Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR–IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163mAmM−1cm−2, a response time less than 6s, and a linear range of 0.25–215μM (R2>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis–Menten constant (KMapp) was calculated to be 2.32mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDs<5%) with minimal interference from the coexisting electroactive compounds such as ascorbic acid and uric acid. The CAT/ChOx/GR–IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples.

Highly Efficient Bienzyme Functionalized Biocompatible Nanostructured Nickel Ferrite–Chitosan Nanocomposite Platform for Biomedical Application

A new hybrid nanocomposite based on hydrothermally synthesized nanostructured NiFe2O4 (n-NiFe2O4) and chitosan (CH) has been explored for bienzyme (cholesterol esterase (ChEt) and cholesterol oxidase (ChOx)) immobilization for application as total cholesterol biosensor. Results of X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), Raman spectroscopy (RS) and vibrating sample magnetometer (VSM) studies demonstrate that the ChEt–ChOx/n-NiFe2O4–CH composite film is successfully synthesized. The obtained ChEt–ChOx/n-NiFe2O4–CH nanocomposite film shows large specific area, high conductivity, good biocompatibility, fast redox properties and improved antimicrobial activity. The fabricated ChEt–ChOx/n-NiFe2O4–CH/ITO bioelectrode exhibits largely improved amperometric biosensing performance, i.e., good linearity (5–400 mg/dL), low detection limit (24.46 mg/dL cm–2), high sensitivity of 1.73 μA/(mg/dL cm–2), fast response time of 15s, reproducibility of more than 15 times, shelf life of about 90 days and low Michaelis–Menten constant (Km) value as 7.05 mg/dL (0.1825 mM). Furthermore, this modified bioelectrode has been utilized for estimation of total cholesterol in human serum samples. This efficient strategy provides new insight into the design of novel flexible electrodes for a wide range of applications in biosensing, bioelectronics, and clinical applications.

Gold nanoparticles: Poly(diallyldimethylammonium chloride)–carbon nanotubes composites as platforms for the preparation of electrochemical enzyme biosensors: Application to the determination of cholesterol

Enzyme biosensors for cholesterol were prepared using a transduction platform constituted of a glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs)/poly-(diallyldimethyl-ammonium chloride) (PDDA)–multi-walled carbon nanotubes (MWCNTs) nanocomposites. This modified electrode exhibited an improved response to H2O2 when compared with other configurations based on CNTs. The incorporation of cholesterol oxidase (ChOx) to this electrode matrix allows the preparation of a biosensor that responded linearly to cholesterol in the 0.02–1.2mM range with a slope of 2.23μA/mM and a limit of detection 4.4μM. All the variables involved in the preparation and performance of the modified electrode and the enzyme biosensor were optimized. The interferences caused by the presence of ascorbic acid and/or uric acid were minimized by coating the electrode surface with a thin film of Nafion which also enhanced the stability of the biosensor. Moreover, the possibility of preparing bienzyme ChOx–HRP biosensors using the same electrode platform was demonstrated. This allowed a detection potential as low as −50mV to be applied using hydroquinone as mediator. With this design, the sensitivity was almost one order of magnitude higher than that achieved with the single ChOx biosensor and no interference from ascorbic acid or uric acid was apparent. Both biosensors designs were used for the determination of cholesterol in human serum samples spiked at physiological levels with recoveries ranging between 98.2% and 104%.

Potassium-doped carbon nanotubes toward the direct electrochemistry of cholesterol oxidase and its application in highly sensitive cholesterol biosensor

We demonstrate herein a newly developed serum total cholesterol biosensor by using the direct electron transfer of cholesterol oxidase (ChOx), which is based on the immobilization of cholesterol oxidase and cholesterol esterase (ChEt) on potassium-doped multi-walled carbon nanotubes (KMWNTs) modified electrodes. The KMWNTs accelerate the electron transfer from electrode surface to the immobilized ChOx, achieving the direct electrochemistry of ChOx and maintaining its bioactivity. As a new platform in cholesterol analysis, the resulting electrode (ChOx/KMWNTs/GCE) exhibits a sensitive response to free cholesterol, with a linear range of 0.050–16.0 μmol L −1 and a detection limit of 5.0 nmol L −1 (S/N = 3). Coimmobilization of ChEt and ChOx (ChEt/ChOx/KMWNTs/GCE) allows the determination of both free cholesterol and esterified cholesterol. The resulting biosensor shows the same linear range of 0.050–16.0 μmol L −1 for free cholesterol and cholesteryl oleate, with the detection limit of 10.0 and 12.0 nmol L −1 (S/N = 3), respectively. The concentrations of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, are in good agreement with those determined by the well-established techniques using the spectrophotometry.

Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples

A novel scheme for the fabrication of gold nanoparticle modified cholesterol oxidase based bioelectrode is presented and its application potential for cholesterol biosensor is investigated. The fabrication procedure is based on the deposition of gold nanoparticles on the 1,6-hexanedithiol modified gold electrode, functionalization of the surface of deposited gold nanoparticles with carboxyl groups using 11-mercaptoundecanoic acid and then covalent immobilization of cholesterol oxidase on the surface of gold nanoparticle film using the N-ethyl-N′-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry. The assembly process of the bioelectrode is investigated using atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticle film on the electrode surface provided an environment for the enhanced electrocatalytic activities and thus resulted in enhanced analytical response. The resulting bioelectrode is further applied to the amperometric detection of cholesterol and exhibited a linear response to cholesterol in the range of 0.04–0.22mM with a detection limit of 34.6μM, apparent Michaelis–Menten constant (Kmapp) of 0.062mM and a high sensitivity of 9.02μAmM−1. The fabricated bioelectrode is successfully used for the selective determination of cholesterol in human serum samples.

Amperometric determination of total cholesterol at gold electrodes covalently modified with cholesterol oxidase and cholesterol esterase with use of thionin as an electron mediator.

Immobilization of cholesterol oxidase (EC 1.1.3.6) (ChOx) on a gold electrode was attempted by cross-linking using glutaraldehyde between ChOx molecules and a self-assembled monolayer of 2-aminoethanethiolate. The resulting electrode (ChOx/Au) exhibits an amperometric response to free cholesterol in the presence of thionin as an electron mediator, and a steady-state response is obtained approximately 60 s after injection of cholesterol into the electrolyte solution. Coimmobilization of cholesterol esterase (EC 3.1.1.13) (ChE) and ChOx (ChE/ChOx/Au) allows the amperometric determination of both esterified cholesterol and free cholesterol. Cyclic voltammetry of the ChE/ChOx/Au and the dependence of the amperometric response to cholesterol on the concentration of thionin suggest that thionin is encapsulated in the enzyme film on the electrode surface. Apparent Michaelis constants of the ChOx/Au and the ChE/ChOx/Au electrodes suggest that the amperometric response was controlled by penetration of the reaction substrate into the films of the enzyme(s). The concentration of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, is in good agreement with that determined by the well-established technique using colorimetry.