Cholesterol Oxidase Research Articles

Aggregation-induced emissive copper nanoclusters with peroxidase-like activity for colorimetric detection of cholesterol

Accurate and point-of-care cholesterol detection is of paramount significance for the prevention of cardiovascular diseases. The colorimetric assay based on peroxidase is a commonly used approach for cholesterol detection, without requiring any complicated biomolecular labeling or sophisticated instrumentation. Copper nanoclusters (CuNCs), exhibiting luminescent properties and peroxidase activity, have garnered significant attention in biomedical application recently. Herein, the glutathione-stabilized copper nanoclusters (GSH-CuNCs) were prepared with an easy one-pot method, employing glutathione as both a reducing agent and stabilizer. An optimization of the GSH-CuNCs preparation was carried out to obtain the highest peroxidase-like activity. UV-Vis absorption was measured to explore the steady-state kinetics of the GSH-CuNCs-catalyzed oxidation of 3,3',5,5' - tetramethylbenzidine (TMB) by H2O2. A colorimetric method for cholesterol detection was developed by combining the catalytic reaction of CuNCs and the enzymatic oxidation of cholesterol with cholesterol oxidase (ChOx). Under the optimized conditions, the UV-Vis absorbance of oxidized TMB (oxTMB) is proportional to the concentration of cholesterol within the range of 6.2-187.5 μM, and the limit of detection (LOD) is determined to be 3.0 μM. More importantly, cholesterol levels can be directly distinguished with the naked eye. Furthermore, the practicality of the method for detecting cholesterol in human serum has been verified with promising results. As expected, this simple, cost-effective, and easy-to-operate colorimetric method for cholesterol detection has potential applications in clinical diagnosis and provides valuable insights into the colorimetric sensing based on CuNCs.

Paper immobilized BSA-decorated gold nanoclusters for single-step optical sensing of glucose and cholesterol without cross-reactivity

Minimally invasive methods for detecting glucose, cholesterol and hydrogen peroxide are crucial for monitoring the nutritional and health status of humans and animals. The peroxidase mimetic activity by nanozymes is one of the versatile methods for detecting glucose, cholesterol, hydrogen peroxide, and other biomolecules. However, the strict requirement of acidic pH limits their sensing and interfacing ability with natural enzymes. The present study developed bovine serum albumin (BSA) coated gold nanoclusters (AuNC) immobilized on paper fabric to enable single-step visual detection of glucose, cholesterol and hydrogen peroxide in complex biological fluids like serum and milk. The BSA-AuNC suspension and immobilized paper fabric synergistically interface with the natural oxidative enzymes, glucose oxidase or cholesterol oxidase, at physiological pH. The concomitant loss in the fluorescent intensity of BSA-AuNC-loaded paper fabric exposed to the generated hydrogen peroxide (glucose/glucose oxidase or cholesterol/cholesterol oxidase) was directly proportional to the concentration of glucose or cholesterol. These reactions enabled simple visual detection as well as quantification of hydrogen peroxide, glucose and cholesterol using Image-J software and common smartphone-based mobile applications. The detection ability of BSA-AuNC-embedded paper fabric is specific and remains unaltered in the presence of similar oxidase enzymes or similar substrate analogues. With these unique features, the BSA-AuNC embedded paper fabric stands out as a prominent analytical device with enormous potential as a simple, user-friendly detection tool for monitoring biomolecules that are important to health, nutrition, and environmental safeguarding.

An improved amperometric cholesterol biosensor based on cholesterol oxidase nanostructures for pre-diagnosis of myocardial infarction

Cholesterol plays a pivotal role in human health, serving as a crucial biomarker for cardiovascular diseases, including myocardial infarction. This study presents the development of an innovative amperometric cholesterol biosensor that enhances the detection and quantification of cholesterol levels in serum. The biosensor integrates cholesterol oxidase (ChOx) nanoparticles with a modified electrode, leveraging the unique properties of platinum nanoparticles (PtNPs) and graphene nanosheets (GNs) to improve sensitivity and stability. The synthesis of PtNPs was achieved using Camellia sinensis extract, while graphene oxide was reduced to form GNs. At 2.39 mg/mL or above is deemed a biomarker for cardiovascular disorders, peripheral artery disease, heart attack, diabetes mellitus, strokes, and hypertension. The monitoring of serum cholesterol level is therefore very significant. In the present study, an innovative amperometric cholesterol biosensor was constructed by immobilizing nanoparticles of cholesterol oxidase onto a pencil graphite (PG) electrode modified with graphene nanosheets (GNs), platinum nanoparticles (PtNPs), and chitosan (CHIT). At various stages of construction, the modified electrode was characterized by employing electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) spectroscopy. The biosensor responded best when run at 0.14Vs-1 at optimal pH and temperature of 8.0 and 35°C respectively. The biosensor has a wide linear range (0.1mg/mL-7.5 mg/mL), with great sensitivity (0.89 mA cm−1mgmL−1) and a low limit of detection (0.97 mg/mL). This research not only contributes to the field of biosensing but also offers a promising tool for the early diagnosis of cholesterol-related health issues, paving the way for enhanced cardiovascular disease management.

Silver shelled gold nanorods for sensitive detection of cholesterol and triglycerides

We report the synthesis of bimetallic plasmonic nanostructure of silver shelled gold nanorods (Ag–Au NRs), subsequently employed for the selective detection of cholesterol (Cho) and triglycerides (TGl) utilizing Surface Enhanced Raman Spectroscopy (SERS). In this direction, the gold nanorods (Au NRs) were synthesized via seed mediated growth method followed by forming a silver shell with reduction of AgNO3 onto Au NRs. Ag–Au NRs showed significantly augmented SERS property owing to hybridization of Localized Surface Plasmon Resonances (LSPR) of silver and gold. The enhanced plasmonic property was thus employed for biosensing. For this, two different Raman reporter molecules, 5,5′–dithiobis–(2–nitrobenzoic acid) (DTNB) and 4–aminothiophenol (4ATP) were immobilized separately on Ag–Au NRs to synthesize SERS active nanoprobes, before the attachment of the bioreceptors – cholesterol oxidase (ChOx) and lipase (Lp) to form ChOx–DTNB–Ag–Au NRs and Lp–4ATP–Ag–Au NRs. These nanoprobes were then utilized for the quantification of Cho and TGl via liquid mode Raman spectroscopic study. The change in SERS spectral intensity of DTNB and 4ATP were systematically recorded in reference to the baseline sample to mark the calibration for both Cho and TGl. Additionally, interference studies considering effects of ascorbic acid, glucose, sodium and potassium ion were performed to unveil excellent selectivity of the proposed method.

Molecular characterization and chemical engineering of cholesterol oxidase from endophytic bacteria in some medicinal plants

Molecular characterization and chemical engineering of cholesterol oxidase from endophytic bacteria in some medicinal plants

Electrodeposited ZnO Nanostructures on ITO Surfaces: Exploring Their Efficacy for Cholesterol Biosensing Applications

In this study, ZnO nanostructures were prepared by electrochemical anodization of electrodeposited Zn on ITO/glass substrates for cholesterol detection. The efficiency of the developed ZnO nanostructures in the detection of the Cholesterol oxidase (ChOx) enzyme was determined by the cyclic voltammetry method. The XRD and SEM results confirmed the synthesis of ZnO nanostructures prepared by the anodization method with various parameters. The effect of electrodeposition and anodization time on the morphology was observed. Cyclic voltammetry of ZnO/Zn/ITO/glass and Pt/ZnO/Zn/ITO/glass electrodes in electrolytes with various cholesterol concentrations was performed. The detection limit of the obtained Pt/ZnO/Zn/ITO/glass structured electrode was calculated as 0.965x10-3M. The resulting material with a layered structure may have potential applications in electrochemical sensors and biosensors in biomedical applications. In addition to biosensing performance, this study proposes a new approach for the development of ZnO-based biosensors that does not require expensive infrastructure and raw material costs, making it possible to develop high-sensitivity biosensor electrodes with lower detection limits with improvements to be made in future studies.

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.

High-efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro-emulsion system.

21-Hydroxy-20-methylpregn-4-en-3-one (4-HBC, bisnoralcohol) is a crucial intermediate for the synthesis of steroidal drugs. Significant challenges including by-products formation and poor substrate solubility were still confronted in its main synthetic route by microbial conversion from phytosterol. Construction of a direct bioconversion pathway to 4-HBC and an efficient substrate emulsion system is therefore urgently required. In this study, three novel isoenzymes of 3-ketosteroid-Δ1-dehydrogenase (KstD) and 3-ketosteroid 9α-hydroxylase (KsH) in Mycobacterium neoaurum were excavated and identified as KstD4, KstD5, and KsHA3. A strain capable of fully directing the synthesis of 4-HBC was metabolically engineered via serial genetic deletion combined with enhanced expression of cholesterol oxidase (ChOx2) and enoyl-CoA hydratase (EchA19). Moreover, a micro-emulsion system combined with soybean oil and hydroxypropyl-β-cyclodextrin improved substrate solubility and bioavailability. In batch fermentation, molar yield of 96.7% with 39.5 gL-1 4-HBC was obtained from 50 gL-1 phytosterol. Our findings demonstrate the potential for industrial-scale biosynthesis of 4-HBC.

Oxidation of lipid membrane cholesterol by cholesterol oxidase and its effects on raft model membrane structure

The effects of a peripheral protein – cholesterol oxidase (3β-hydroxysteroid oxidase, ChOx) on the characteristics of model lipid membranes composed of cholesterol, cholesterol:sphingomyelin (1:1), and the raft model composed of DOPC:Chol:SM (1:1:1) were investigated using two membrane model systems: the flat monolayer prepared by the Langmuir technique and the curved model consisting of liposome of the same lipids. The planar monolayers and liposomes were employed to follow membrane cholesterol oxidation to cholestenone catalyzed by ChOx and changes in the lipid membrane structure accompanying this reaction. Changes in the structure of liposomes in the presence of the enzyme were reflected in the changes of hydrodynamic diameter and fluorescence microscopy images, while changes of surface properties of planar membranes were evaluated by grazing incidence X-ray diffraction (GIXD) and Brewster angle microscopy. UV-Vis absorbance measurements confirmed the activity of the enzyme in the tested systems. A better understanding of the interactions between the enzyme and the cell membrane may help in finding alternative ways to decrease excessive cholesterol levels than the common approach of treating hypercholesterolemia with statins, which are not free from undesirable side effects, repeatedly reported in the literature and observed by the patients.

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.

Cholesterol Depletion-Enhanced Ferroptosis and Immunotherapy via Engineered Nanozyme.

Ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, holds significant promise for tumor therapy due to its ability to induce lipid peroxidation (LPO) and trigger antitumor immune responses. However, elevated cholesterol levels in cancer cells impede ferroptosis and compromise immune function. Here, a novel nanozyme, Fe-MOF/CP, composed of iron metal-organic framework (Fe-MOF) nanoparticles loaded with cholesterol oxidase and PEGylation for integrated ferroptosis and immunotherapy is introduced. Fe-MOF/CP depletes cholesterol and generates hydrogen peroxide, enhancing ROS levels and inducing LPO, thereby promoting ferroptosis. This process disrupts lipid raft integrity and downregulates glutathione peroxidase 4 and ferroptosis suppressor protein 1, further facilitating ferroptosis. Concurrently, Fe-MOF/CP augments immunogenic cell death, reduces programmed death-ligand 1 expression, and revitalizes exhausted CD8+ T cells. In vivo studies demonstrate significant therapeutic efficacy in abscopal, metastasis, and recurrent tumor models, highlighting the robust antitumor immune responses elicited by Fe-MOF/CP. This study underscores the potential of Fe-MOF/CP as a multifunctional therapeutic agent that combines ferroptosis and immunotherapy, offering a promising strategy for effective and durable cancer treatment.

Enzyme Engineering to Produce SWCNT Nanosensors as a Generalizable Platform for Biochemical Imaging and Sensing

Single-walled carbon nanotubes (SWCNT) stand out as attractive candidates for near-infrared (nIR) fluorescent imaging and chemical sensing. However, to render SWCNT chemically-selective, rational design of the surface corona is required to achieve sufficient sensitivity and selectivity to the target analyte. Recently, we reported on a facile sonication-based generation of glucose oxidase (GOx)-SWCNT conjugate for glucose detection [1]. Notably, we found that the analyte response mechanism does not involve catalytic oxidation of glucose, which motivated us to develop nanosensors using catalytically inactive GOx for enhanced reversibility and biocompatibility. In this study, we study the generalizability of our approach in producing SWCNT-based nanosensors based on 1- facile direct probe tip sonication, and 2- with engineered enzymes, showing the potential applicability of the sonication-based approach as a generalizable strategy for generating rationally designed nanosensors capable of detecting and imaging various analytically-relevant biomolecules.In this work, to test the generalizability of generating enzyme-SWCNT nanosensors, we generated conjugates by facile direct sonication of SWCNT with following enzymes: GOx, choline oxidase (ChOx), horseradish peroxidase (HRP), acetylcholineesterase, tyrosinase, cholesterol oxidase (CholOx), lactate oxidase, alcohol oxidase, xanthine oxidase, and galactose oxidase. Remarkably, six out of nine enzymes yielded stable suspensions of enzyme-SWCNT conjugates following probe-tip sonication, all of which successfully worked as nanosensors to detect their corresponding analytes. These nanosensors, in particular HRP-SWCNT and CholOx-SWCNT, exhibited excellent responses to their targets, with a maximum ΔF/F 0 of up to 300 % and 100 %, respectively. H2O2 is known to quench SWCNT fluorescence, and H2O2 nanosensors previously developed with SWCNT exhibited a negative fluorescence modulation as the sensor output with some challenges towards nanosensor selectivity. We highlight that our approach enables HRP-SWCNT nanosensor generation, which provides a strong ΔF/F 0 = 300 % and instantaneous turn-on response towards H2O2 within 2 s. Next, we sought to show that enzyme inactivation can produce SWCNT-based nanosensors that detect analytes without analyte consumption. To do so, we use ChOx as a model system to show that recombinant enzymes can be used as a generalizable approach to generating catalytically-inactive nanosensors. As expected, nanosensors prepared with mutant-ChOx exhibited responses comparable to those prepared with native ChOx, but without consuming the analyte or producing toxic byproducts such as H2O2. Thus, sonication-based physisorption of engineered enzymes to SWCNTs holds the potential to facilitate rapid nanosensor generation capable of detecting various biologically relevant molecules with reversibility and biocompatibility, thereby motivating their use in in vivo applications.[1] Nishitani, S., Tran, T., Puglise, A., Yang, S. & Landry, M. Engineered Glucose Oxidase-Carbon Nanotube Conjugates for Tissue-Translatable Glucose Nanosensors. Angew. Chem. Int. Ed Engl. e202311476 (2023)

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.

B, N co-doped carbon dots as efficient nanozymes for colorimetric and fluorometric dual-mode detection of cholesterol

In this work, the B, N co-doped carbon dots (B, N-CDs) were synthesized via facile hydrothermal approach with 6-aminopyridine boronic acid as precursor. In addition to emitting intense blue luminescence when exposed to ultraviolet light, the prepared B, N-CDs displayed remarkable peroxidase-like activity, which could efficiently catalyze the oxidation of 3, 3′, 5, 5′ -tetramethylbenzidine (TMB) to blue ox-TMB in the presence of hydrogen peroxide (H2O2). Furthermore, the fluorescence intensity of B, N-CDs increased gradually upon the addition of H2O2. Since cholesterol oxidase (ChOx) can catalyze the oxidation of cholesterol to form H2O2, the as-prepared B, N-CDs was then used as both colorimetric and fluorometric sensors for the detection of cholesterol with detection limit of 0.87 and 2.31 μM, respectively. Finally, the dual-mode approach based on B, N-CDs was effectively utilized for detecting cholesterol levels in serum samples, proving the potential application of B, N-CDs in the field of biological assay.

Effects of membrane cholesterol-targeting chemicals on skeletal muscle contractions evoked by direct and indirect stimulation.

Cholesterol is one of the major components of plasma membrane, where its distribution is nonhomogeneous and it participates in lipid raft formation. In skeletal muscle cholesterol and lipid rafts seem to be important for excitation-contraction coupling and for neuromuscular transmission, involving cholesterol-rich synaptic vesicles. In the present study, nerve and muscle stimulation-evoked contractions were recorded to assess the role of cholesterol in contractile function of mouse diaphragm. Exposure to cholesterol oxidase (0.2 U/ml) and cholesterol-depleting agent methyl-β-cyclodextrin (1 mM) did not affect markedly contractile responses to both direct and indirect stimulation at low and high frequency. However, methyl-β-cyclodextrin at high concentration (10 mM) strongly decreased the force of both single and tetanus contractions induced by phrenic nerve stimulation. This decline in contractile function was more profoundly expressed when methyl-β-cyclodextrin application was combined with phrenic nerve activation. At the same time, 10 mM methyl-β-cyclodextrin had no effect on contractions upon direct muscle stimulation at low and high frequency. Thus, strong cholesterol depletion suppresses contractile function mainly due to disturbance of the neuromuscular communication, whereas muscle fiber contractility remains resistant to decline.

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.

MOF-derived nanozyme CuOx@C and its application for cascade colorimetric detection of phytosterols.

Phytosterols (PSs), a class of naturally occurring bioactive lipid compounds, have been found to possess a significant cholesterol-lowering effect. In developing countries, the consumption of rapeseed oil is the primary pathway of PS intake for the general population. However, developing low-cost, real-time, and high-throughput screening techniques for PSs remains a challenge. Here, a Cu-based nanocomposite CuOx@C was synthesized via a simple method of the calcination of HKUST-1 and systematically characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The CuOx@C demonstrated excellent peroxidase-like (POD-like) activity, functioning as a peroxidase mimic to facilitate the catalysis of 3,3',5,5'-tetramethylbenzidine (TMB) into its oxidized form (oxTMB), thereby initiating a discernible color response. On the basis of this discovery, a CuOx@C-based colorimetric method for detecting total sterols in rapeseed was successfully constructed via cascade reactions. After optimizing the conditions, the high-throughput screening of total sterols in rapeseed could be completed in only 21min, which significantly facilitated the sensing of PSs. A linear range of 0.6-6mg/g was achieved for the detection of total sterols in rapeseed samples, thereby satisfying the requirements for detection. In addition, due to the high stability of CuOx@C and the specificity of cholesterol oxidase, the developed method had excellent stability and selectivity toward PSs, indicating that this work has huge prospects for commercial application. This innovative work overcomes the limitation of the instrumental method and provides a portable and reliable tool for total sterols detection. It can also facilitate the development of oilseeds with a high content of PSs.

The role of cholesterol and its oxidation products in tuberculosis pathogenesis.

Mycobacterium tuberculosis causes tuberculosis (TB), one of the world's most deadly infections. Lipids play an important role in M. tuberculosis pathogenesis. M. tuberculosis grows intracellularly within lipid-laden macrophages and extracellularly within the cholesterol-rich caseum of necrotic granulomas and pulmonary cavities. Evolved from soil saprophytes that are able to metabolize cholesterol from organic matter in the environment, M. tuberculosis inherited an extensive and highly conserved machinery to metabolize cholesterol. M. tuberculosis uses this machinery to degrade host cholesterol; the products of cholesterol degradation are incorporated into central carbon metabolism and used to generate cell envelope lipids, which play important roles in virulence. The host also modifies cholesterol by enzymatically oxidizing it to a variety of derivatives, collectively called oxysterols, which modulate cholesterol homeostasis and the immune response. Recently, we found that M. tuberculosis converts host cholesterol to an oxidized metabolite, cholestenone, that accumulates in the lungs of individuals with TB. M. tuberculosis encodes cholesterol-modifying enzymes, including a hydroxysteroid dehydrogenase, a putative cholesterol oxidase, and numerous cytochrome P450 monooxygenases. Here, we review what is known about cholesterol and its oxidation products in the pathogenesis of TB. We consider the possibility that the biological function of cholesterol metabolism by M. tuberculosis extends beyond a nutritional role.

An in situ one-step route for the synthesis of Fe3O4@ZIF-8 core-shells with a high-peroxidase-like performance for the colorimetric detection of methyl parathion

Methyl parathion (MP) is a broad-spectrum agricultural insecticide. Human exposure to MP can affect the nervous, cardiovascular, and hepatic systems. In this research, a core–shell Fe3O4@zeolitic imidazolate framework-8 (ZIF-8) is developed as a peroxidase mimic for the rapid and simple colorimetric detection of MP. Fe3O4 nanoparticles were wrapped with ZIF-8 by a hydrothermal method to obtain core–shell structural Fe3O4@ZIF-8 nanocomposites. The synthesized Fe3O4@ZIF-8 possessed mimetic properties and effectively catalyzed the conversion of transparent solution to blue upon the oxidation of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) by H2O2. Further, based on a classical reaction through acetylthiocholine chloride (ATCl) and cholesterol oxidase (ChOx), acetylcholinesterase (AChE) can generate H2O2, and that MP can exert an inhibitory effect on AChE. Within the range of 10 to 200 µg/L, the MP concentration demonstrated a remarkable linear response with the absorbance under optimum conditions, with a detection limit of 6.3 μg/L. Finally, the proposed approach enabled the detection of MP in cabbage. As an artificial peroxidase, Fe3O4@ZIF-8 demonstrated greater affinity for the substrate than horseradish peroxidase, which has a great application prospects in food safety and environmental safety.

A hybrid catalyst-triggered cascade reaction for cholesterol detection using a smartphone-based miniature fluorescent apparatus

A new hybrid biological-chemical catalyst, magnetic nanoparticles functionalized with cholesterol oxidase (Fe3O4/APTES/ChOx), was developed for cholesterol detection. In the presence of cholesterol, the enzyme produced H2O2, which facilitated the generation of fluorescent molecules from the fluorogenic substrate with the assistance of Fe3O4 nanoparticles. A smartphone camera with a miniature fluorescent apparatus was used to assess fluorescence emission. Then, a smartphone application was employed to translate the fluorescence intensity to the red, green, and blue (RGB) domain. The developed approach achieved excellent selectivity and acceptable performances while supporting an onsite analysis approach. The practical operational range spanned from 5 to 100 nM, with a detection limit of 0.85 nM. Fe3O4/APTES/ChOx was applied for up to four replicates of reuse and demonstrated stability for at least 30 days. The applicability of the method was evaluated in milk samples, and the results were in accordance with the reference method.