The importance of choosing the appropriate cholesterol quantification method: enzymatic assay versus gas chromatography.

Cholesterol is a major lipid of the animal realm with many biological roles. It is an important component of cellular membranes and a precursor of steroid hormones and bile acids. It is particularly abundant in nervous tissues, and dysregulation of cholesterol metabolism has been associated with neurodegenerative diseases such as Alzheimer's and Huntington's diseases. Deciphering the pathophysiological mechanisms of these disorders often involves animal models such as mice and Drosophila. Accurate quantification of cholesterol levels in the chosen models is a critical point of these studies. In the present work, we compare two common methods, gas chromatography coupled to flame-ionization detection (GC/FID) and a cholesterol oxidase-based fluorometric assay to measure cholesterol in mouse brains and Drosophila heads. Cholesterol levels measured by the two methods were similar for the mouse brain, which presents a huge majority of cholesterol in its sterol profile. On the contrary, depending on the method, measured cholesterol levels were very different for Drosophila heads, which present a complex sterol profile with a minority of cholesterol. We showed that the enzyme-based assay is not specific for cholesterol and detects other sterols as well. This method is therefore not suited for cholesterol measurement in models such as Drosophila. Alternatively, chromatographic methods, such as GC/FID, offer the required specificity for cholesterol quantification. Understanding the limitations of the quantification techniques is essential for reliable interpretation of the results in cholesterol-related research.

Multi-Enzyme Cascade Nanoreactors for High-Throughput Immunoassay: Transitioning Concept in Lab to Application in Community.

In this work, we reported a cholesterol oxidase (Chox)-loaded platinum (Pt) nanozyme with the collaborative cascade nanoreactor for the construction of nanozyme-enzyme-linked immunosorbent assay (N-ELSA) models to realize high-throughput rapid evaluation of cancer markers. Considering the high specific surface area and manipulable surface sites, ZIF-8 was used as a substrate for natural enzyme and nanozyme loading. The constructed ZIF-8-Pt nanozyme platform exhibited efficient enzyme-like catalytic efficiency with a standard corrected activity of 60.59 U mg-1, which was 12 times higher than that of the ZIF-8 precursor, and highly efficient photothermal conversion efficiency (∼35.49%). In N-ELISA testing, developed multienzyme photothermal probes were immobilized in microplates based on antigen-antibody-specific reactions. Cholesterol was reacted in a cascade to reactive oxygen radicals, which attacked 3,3',5,5'-tetramethylbenzidine, causing it to oxidize and color change, thus exhibiting highly enhanced efficient photothermal properties. Systematic temperature evaluations were performed by a hand-held microelectromechanical system thermal imager under the excitation of an 808 nm surface light source to determine the cancer antigen 15-3 (CA15-3) profiles in the samples. Encouragingly, the temperature signal from the microwells increased with increasing CA15-3, with a linear range of 2 mU mL-1 to 100 U mL-1, considering it to be the sensor with the widest working range for visualization and portability available. This work provides new horizons for the development of efficient multienzyme portable colorimetric-photothermal platforms to help advance the community-based process of early cancer detection.

Enhanced effectiveness in preventing Nocardia seriolae infection utilizing heterologous prime-boost approach in orange-spotted grouper Epinephelus coioides.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine's efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

Hive-shaped carbon nanotubes coated with MoSe2as an electrochemical sensor for cholesterol.

The study utilized transition metal chalcogenide, molybdenum diselenide (MoSe2), for application in the field of bioelectrochemical sensing. The MoSe2was combined with carbon nanotubes (CNTs) by chemical vapor deposition to enhance the specific surface area and improve the detection sensitivity. To further increase the contact area between the electrolyte and the electrode, photolithography techniques were employed to fabricate hive-shaped CNTs, thereby enhancing the specific surface area. Next, cholesterol oxidase (ChOx) was coated onto the electrode material, creating a cholesterol biosensor. Cyclic voltammetry was utilized to detect the concentration of cholesterol. The experiment involved segmented testing for cholesterol concentrations ranging from 0µM to 10 mM. Excellent sensitivity, low detection limits, and high accuracy were achieved. In the cholesterol concentration range of 0µM-100µM, the experiment achieved the highest sensitivity of 4.44µAµM⋅cm-2. Consequently, all data indicated that ChOx/MoSe2/CNTs functioned as an excellent cholesterol sensor in the study.

Mechanoregulatory Cholesterol Oxidase-Functionalized Nanoscale Metal-Organic Framework Stimulates Pyroptosis and Reinvigorates T Cells.

Cancer cells alter mechanical tension in their cell membranes. New interventions to regulate cell membrane tension present a potential strategy for cancer therapy. Herein, the increase of cell membrane tension by cholesterol oxidase (COD) via cholesterol depletion in vitro and the design of a COD-functionalized nanoscale metal-organic framework, Hf-TBP/COD, for cholesterol depletion and mechanoregulation of tumors in vivo, are reported. COD is found to deplete cholesterol and disrupt the mechanical properties of lipid bilayers, leading to decreased cell proliferation, migration, and tolerance to oxidative stress. Hf-TBP/COD increases mechanical tension of plasma membranes and osmotic fragility of cancer cells, which induces influx of calcium ions, inhibits cell migration, increases rupturing propensity for effective caspase-1 mediated pyroptosis, and decreases tolerance to oxidative stress. In the tumor microenvironment, Hf-TBP/COD downregulates multiple immunosuppressive checkpoints to reinvigorate T cells and enhance T cell infiltration. Compared to Hf-TBP, Hf-TBP/COD improves anti-tumor immune response and tumor growth inhibition from 54.3% and 79.8% to 91.7% and 95% in a subcutaneous triple-negative breast cancer model and a colon cancer model, respectively.

Effects of cholesterol oxidase on neurotransmission and acetylcholine levels at the mice neuromuscular junctions.

Membrane cholesterol oxidation is a hallmark of redox and metabolic imbalance, and it may accompany neurodegenerative disorders. Using microelectrode recordings of postsynaptic responses as well as fluorescent dyes for monitoring synaptic vesicle cycling and membrane properties, the action of enzymatic cholesterol oxidation on neuromuscular transmission was studied in the mice diaphragms. Cholesterol oxidase (ChO) at low concentration disturbed lipid-ordering specifically in the synaptic membranes, but it did not change markedly spontaneous exocytosis and evoked release in response to single stimuli. At low external Ca2+ conditions, analysis of single exocytotic events revealed a decrease in minimal synaptic delay and the probability of exocytosis upon plasmalemmal cholesterol oxidation. At moderate- and high-frequency activity, ChO treatment enhanced both neurotransmitter and FM-dye release. Furthermore, it precluded a change in exocytotic mode from full-fusion to kiss-and-run during high-frequency stimulation. Accumulation of extracellular acetylcholine (without stimulation) dependent on vesamicol-sensitive transporters was suppressed by ChO. The effects of plasmalemmal cholesterol oxidation on both neurotransmitter/dye release at intense activity and external acetylcholine levels were reversed when synaptic vesicle membranes were also exposed to ChO (i.e., the enzyme treatment was combined with induction of exo-endocytotic cycling). Thus, we suggest that plasmalemmal cholesterol oxidation affects exocytotic machinery functioning, enhances synaptic vesicle recruitment to the exocytosis and decreases extracellular neurotransmitter levels at rest, whereas ChO acting on synaptic vesicle membranes suppresses the participation of the vesicles in the subsequent exocytosis and increases the neurotransmitter leakage. The mechanisms underlying ChO action can be related to the lipid raft disruption.

An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides.

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1ß, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.

Facile immobilization of cholesterol oxidase on Pt,Ru-C nanocomposite and ionic liquid-modified carbon paste electrode for an efficient amperometric free cholesterol biosensing.

In present work, the enzyme cholesterol oxidase (ChOx) was immobilized by Nafion® (Naf) on Pt,Ru-C nanocomposite and an ionic liquid (IL)-modified carbon paste electrode (CPE) in order to create cholesterol biosensor (Naf/ChOx/Pt,Ru-C/IL-CPE). The prepared working electrodes were characterized using scanning electron microscopy-energy-dispersive spectrometry, while their electrochemical performance was evaluated using electrochemical impedance spectroscopic, cyclic voltammetric, and amperometric techniques. Excellent synergism between IL 1-allyl-3-methylimidazolium dicyanamide ([AMIM][DCA]), Pt,Ru-C, and ChOx, as modifiers of CPE, offers the most pronounced analytical performance for improved cholesterol amperometric determination in phosphate buffer solution pH 7.50 at a working potential of 0.60 V. Under optimized experimental conditions, a linear relationship between oxidation current and cholesterol concentration was found for the range from 0.31 to 2.46 µM, with an estimated detection limit of 0.13 µM and relative standard deviation (RSD) below 5.5%. The optimized amperometric method in combination with the developed Naf/ChOx/Pt,Ru-C/IL-CPE biosensor showed good repeatability and high selectivity towards cholesterol biosensing. The proposed biosensor was successfully applied to determine free cholesterol in a human blood serum sample via its enzymatic reaction product hydrogen peroxide despite the presence of possible interferences. The percentage recovery ranged from 99.08 to 102.81%, while RSD was below 2.0% for the unspiked as well as the spiked human blood serum sample. The obtained results indicated excellent accuracy and precision of the method, concluding that the developed biosensor can be a promising alternative to existing commercial cholesterol tests used in medical practice.

Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR.

Cholesterol oxidase is industrially important as it is frequently used as a biosensor in food and agriculture industries and measurement of cholesterol. Although, most natural enzymes show low thermostability, which limits their application. Here, we obtained an improved variant of Chromobacterium sp. DS1 cholesterol oxidase (ChOS) with enhanced thermostability by random mutant library applying two forms of error-prone PCR (serial dilution and single step). Wild-type ChOS indicated an optimal temperature and pH of 70 ºC and pH 7.5, respectively. The best mutant ChOS-M acquired three amino acid substitutions (S112T, I240V and A500S) and enhanced thermostability (at 50 °C for 5 h) by 30%. The optimum temperature and pH in the mutant were not changed. In comparison to wild type, circular dichroism disclosed no significant secondary structural alterations in mutants. These findings show that error-prone PCR is an effective method for enhancing enzyme characteristics and offers a platform for the practical use of ChOS as a thermal-resistance enzyme in industrial fields and clinical diagnosis.

An integrated microfluidic-based biosensor using a magnetically controlled MNPs-enzyme microreactor to determine cholesterol in serum with fluorometric detection.

This work provides a microfluidic-based biosensor to determine total cholesterol in serum based on integrating the reaction/detection zone of a microfluidic chip of a magnetically retained enzyme microreactor (MREµR) coupled with the remote fluorometric detection through a bifurcated fiber-optic bundle (BFOB) connected with a conventional spectrofluorometer. The method is based on developing the enzymatic hydrolysis and oxidation of cholesterol at microscale size using both enzymes (cholesterol esterase (ChE) and cholesterol oxidase (ChOx)) immobilized on magnetic nanoparticles (MNPs). The biocatalyst reactions were followed by monitoring the fluorescence decreasing by the naphtofluorescein (NF) oxidation in the presence of the previous H2O2 formed. This microfluidic biosensor supposes the physical integration of a minimal MREµR as a bioactive enzyme area and the focused BFOB connected with the spectrofluorometer detector. The MREµR was formed by a 1 mm length of magnetic retained 2:1 ChE-MNP/ChOx-MNP mixture. The dynamic range of the calibration graph was 0.005-10 mmol L-1, expressed as total cholesterol concentration with a detection limit of 1.1 µmol L-1 (r2 = 0.9999, sy/x = 0.03, n = 10, r = 3). The precision expressed as the relative standard deviation (RSD%) was between 1.3 and 2.1%. The microfluidic-based biosensors showed a sampling frequency estimated at 30 h-1. The method was applied to determine cholesterol in serum samples with recovery values between 94.8 and 102%. The results of the cholesterol determination in serum were also tested by correlation with those obtained using the other two previous methods.

Expression and characterization of cholesterol oxidase with high thermal and pH stability from Janthinobacterium agaricidamnosum.

Cholesterol oxidases (COXases) have a diverse array of applications including analysis of blood cholesterol levels, synthesis of steroids, and utilization as an insecticidal protein. The COXase gene from Janthinobacterium agaricidamnosum was cloned and expressed in Escherichia coli. The purified COXase showed an optimal temperature of 60 °C and maintained about 96 and 72% of its initial activity after 30 min at 60 and 70 °C, respectively. In addition, the purified COXase exhibited a pH optimum at 7.0 and high pH stability over the broad pH range of 3.0-12.0. The pH stability of the COXase at pH 12.0 was higher than that of highly stable COXase from Chromobacterium sp. DS-1. The COXase oxidized cholesterol and ß-cholestanol at higher rates than other 3ß-hydroxysteroids. The Km, Vmax, and kcat values for cholesterol were 156 µM, 13.7 µmol/min/mg protein, and 14.4 s-1, respectively. These results showed that this enzyme could be very useful in the clinical determination of cholesterol in serum and the production of steroidal compounds. This is the first report to characterize a COXase from the genus Janthinobacterium.

An Engineered Cholesterol Oxidase Catalyses Enantioselective Oxidation of Non-steroidal Secondary Alcohols.

The enantioselective oxidation of 2° alcohols to ketones is an important reaction in synthetic chemistry, especially if it can be achieved using O2 -driven alcohol oxidases under mild reaction conditions. However to date, oxidation of secondary alcohols using alcohol oxidases has focused on activated benzylic or allylic substrates, with unactivated secondary alcohols showing poor activity. Here we show that cholesterol oxidase (EC 1.1.3.6) could be engineered for activity towards a range of aliphatic, cyclic, acyclic, allylic and benzylic secondary alcohols. Additionally, since the variants demonstrated high (S)-selectivity, deracemisation reactions were performed in the presence of ammonia borane to obtain enantiopure (R)-alcohols.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Improvement of thermostability of cholesterol oxidase from streptomyces Sp. SA-COO by random mutagenesis.

To enhance the thermal stability of Streptomyces Sp. SA-COO cholesterol oxidase, random mutagenesis was used. A random mutant library was generated using two types of error-prone PCR (single step and serial dilution) and two mutants (ChOA-M1 and ChOA-M2) with improved thermostability were obtained. The best mutant ChOA-M1 acquired three amino acid substitutions (G49T, W52K, and F62V) and improved thermostability (at 50 °C for 5 h) by 40% and increased the kcat/Km value by 23%. The optimum pH was desirably changed to encompass a broad range from alkali to acid and circular dichroism revealed no significant secondary structure changes in mutants against wild type. These findings indicated that random mutagenesis was an effective technique for optimizing cholesterol oxidase properties and make a foundation for practical applications of Cholesterol oxidase in clinical diagnosis and industrial fields.

A sensitive cholesterol electrochemical biosensor based on biomimetic cerasome and graphene quantum dots.

A simple and sensitive electrochemical cholesterol biosensor was fabricated based on ceramic-coated liposome (cerasome) and graphene quantum dots (GQDs) with good conductivity. The cerasome consists of a lipid-bilayer membrane and a ceramic surface as a soft biomimetic interface, and the mild layer-by-layer self-assembled method as the immobilization strategy on the surface of the modified electrode was used, which can provide good biocompatibility to maintain the biological activity of cholesterol oxidase (ChOx). The GQDs promoted electron transport between the enzyme and the electrode more effectively. The structure of the cerasome-forming lipid was characterized by Fourier transform infrared (FT-IR). The morphology and characteristics of the cerasome and GQDs were characterized by transmission electron microscopy (TEM), zeta potential, photoluminescence spectra (PL), etc. The proposed biosensors revealed excellent catalytic performance to cholesterol with a linear concentration range of 16.0 × 10-6-6.186 × 10-3 mol/L, with a low detection limit (LOD) of 5.0 × 10-6 mol/L. The Michaelis-Menten constant (Km) of ChOx was 5.46 mmol/L, indicating that the immobilized ChOx on the PEI/GQDs/PEI/cerasome-modified electrode has a good affinity to cholesterol. Moreover, the as-fabricated electrochemical biosensor exhibited good stability, anti-interference ability, and practical application for cholesterol detection.

Improving the sensitivity for hydrogen peroxide determination with active V2O5 nanocubes incorporated on mesoporous TiO2.

The capacity to generate a constant signal response from an enzyme on an electrode surface has been a fascinating topic of research from the past three decades. To nourish the enzymatic activity during electrochemical reactions, the immobilization of dual enzymes on the electrode surface could prevent the enzymatic loss without denaturation and thus long-term stability can be achieved. For effective immobilization of dual enzymes, mesoporous materials are the ideal choice because of its numerous advantages such as 1. The presence of porous structure facilitates high loading of enzymes 2. The formation of protective environment can withstand the enzymatic activity even at acidic or basic pH values and even at elevated temperatures. Herein, we develop bienzymatic immobilization of horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) on mesoporous V2O5-TiO2 based binary nanocomposite for effective sensing of hydrogen peroxide (H2O2) in presence of redox mediator hydroquinone (HQ). The utilization of redox mediator in second-generation biosensing of H2O2 can eliminate the interference species and reduces the operating potential with higher current density for electrochemical reduction reaction. Using this mediator transfer process approach at HRP/ChOx/V2O5-TiO2 modified GC, the H2O2 can be determined at operating potential (-0.2 V) with good linear range (0.05-3.5 mM) higher sensitivity (1040 µAµM-1 cm-2) and lower detection limit of about 20 µM can be attained, which is due to higher mediation of electrons were transferred to the enzyme cofactors. These interesting characteristics could be due to mesoporous structure of V2O5-TiO2 can induce large immobilization and facilitate higher interaction with enzymes for wide range of biosensing applications.

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3ß-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

Highly dispersive AuNCs/ChOx@ZIF-8/PEI nanocomplexes for fluorescent detection of cholesterol in human serum.

Gold nanoclusters (AuNCs) are widely used in the fluorescence detection of biomolecules in human serum due to their good fluorescence properties, low toxicity, and better biocompatibility. However, the weak fluorescence intensity of AuNCs limits the fluorescence detection of molecules within a wide concentration range. It is reported that coating AuNCs in ZIF-8 with adjustable pore size can effectively improve the fluorescence intensity of AuNCs and broaden the detection range. And the AuNCs wrapped in the gaps of ZIF-8 can prevent the fluorescence quenching caused by the aggregation of AuNCs. However, ZIF-8 has high crystallinity, poor dispersion, and easy deposition, which reduces the fluorescence stability of the detection system and affects the detection. Based on the above research, the highly hydrophilic polymer PEI was modified to the surface of ZIF-8, and a kind of nanocomposite material AuNCs/ChOx@ZIF-8/PEI was obtained by co-encapsulating AuNCs prepared with glutathione as a ligand and cholesterol oxidase (ChOx) into ZIF-8 modified with PEI. The composite material emits strong red light at 650 nm under the excitation of 395-nm light, and the system can sensitively detect cholesterol (Chol) in human serum. Compared with other materials, the PEI-modified composite has better solubility and stability, so the detection effect of Chol is better. Encapsulation of ChOx in the ZIF-8 shell can protect the enzyme and increase the local concentration of ChOx, thereby speeding up the reaction rate. Compared with free AuNCs/ChOx, the quenching rate of AuNCs/ChOx@ZIF-8/PEI system is doubled. Secondly, the addition of Fe2+ to the detection process results in higher quenching rate and detection sensitivity. The system can detect Chol in the concentration range 0.1-2.4 µM, with a detection limit of 0.073 µM. The determination is a fast and sensitive strategy. In addition, the practicability of this assay in the detection of Chol in human serum has been verified. Due to its selectivity and sensitivity, it has potential application value in clinical diagnosis.

A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

MOF@MnO2nanocomposites prepared usingin situmethod and recyclable cholesterol oxidase-inorganic hybrid nanoflowers for cholesterol determination.

In this work, through thein situgrowth of MnO2nanosheets on the surface of terbium metal-organic frameworks (Tb-MOFs), MOF@MnO2nanocomposites are prepared and the fluorescence of Tb-MOFs is quenched significantly by MnO2. Additionally, the hybrid nanoflowers are self-assembled by cholesterol oxidase (ChOx) and copper phosphate (Cu3(PO4)2·3H2O). Then a new strategy for cholesterol determination is developed based on MOF@MnO2nanocomposites and hybrid nanoflowers. Cholesterol is oxidized under the catalysis of hybrid nanoflowers to yield H2O2, which further reduces MnO2nanosheets into Mn2+. Hence, the fluorescence recovery of Tb-MOFs is positively correlated to the concentration of cholesterol in the range of 10 to 360µM. The limit of detection (LOD) of cholesterol is 1.57µM. On the other hand, the hierarchical and confined structure of ChOx-inorganic hybrid nanoflowers greatly improve the stability of the enzyme. The activity of hybrid nanoflowers remains at a high level for one week when stored at room temperature. Moreover, the hybrid nanoflowers can be collected by centrifugation and reused. The activity of hybrid nanoflowers can continue at a high level for five cycles of determination. Therefore, it can be concluded that the hybrid nanoflowers are more stable and more economic than free enzymes, and they show a similar sensitivity and specificity to cholesterol compared with free ChOx. Finally, this strategy has been further validated for the determination of cholesterol in serum samples with satisfactory recoveries.