Product Of Enzymatic Reaction Research Articles

Allosteric regulation of thioesterase superfamily member 1 by lipid sensor domain binding fatty acids and lysophosphatidylcholine

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.

Inflammatory biomarker detection in milk using label-free porous SiO2 interferometer

N-acetyl-β-d-glucosaminidase (NAGase) is an established indicative biomarker released upon damage or necrosis of tubular epithelial cells in both humans and animals, indicating severe nephrological disorders and bovine mastitis (BM), respectively. The latter is the most common and costly disease in dairy cattle associated with production losses, elevated somatic cell counts and deteriorated health status. Herein, we report on a reflective based assay for early diagnosis of BM through the analysis of NAGase inherent content found in whole milk samples using a miniaturized optical transducer. Gelatin functionalized porous Si Fabry–Pérot interferometers are employed for monitoring the lysosomal activity in various stages of the inflammation (healthy, subclinical and clinical). The enzymatic reaction products precipitate and accumulate within the porous nanostructure, thus alter the average refractive index monitored using reflectometric interference spectroscopy. The optical assay is calibrated within the clinically relevant concentrations of BM while presenting a dynamic range of 1.04–16.7 μM min−1 and the detection limit of 0.49 μM min−1. The specific optical performance of the biosensor correlates with a gold standard laboratory-based approach, in which escalated somatic cell counts reveal augmented NAGase levels and thus severe pathogenesis. Overall, our study provides new opportunities to develop a convenient bio-diagnostic sensing system for BM detection and classification by addressing the limitations of conventional practices.

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Sensing platform for pico-molar level detection of ethyl parathion using Au–Ag nanoclusters based enzymatic strategy

This work demonstrates a simple, cost effective and ultrasensitive detection of ethyl parathion, an organophosphorus (OPs) pesticide, using enzyme based fluorometric sensing strategy by employing bimetallic BSA@AuAg nanoclusters (NC). The sensing assay is based on the “quenched off” state of bimetallic NC with the addition of Cu2+ ions that can be “switched on” due to generation of thiocholine (TCh), a catalytic product of enzymatic reaction of acetylthiocholine (ATCh) using acetylcholinesterase (AChE) enzyme. The generated TCh preferably seize Cu2+ ions from BSA@AuAg NC-Cu2+ ensemble and recovered the fluorescence of BSA@AuAg NC. The presence of ethyl parathion can be monitored optically due to its inhibitory action towards AChE enzyme leading to suppression of thiocholine (TCh) formation and subsequently decreases TCh-Cu2+ interaction that ultimately retrieved quenched off state of bimetallic NC. The synthesized biosensor is appropriate for the ultrasensitive sensing of ethyl parathion in pM range, exhibiting 2.40 pM as lowest limit of detection (LOD) which is the least known so far. Further, the real sample analysis adds on for the appropriateness of the synthesized nanoprobe by depicting excellent reproducibility and robustness. The designed assay proved its specificity towards pesticides in general and ethyl parathion in particular when employed with other commonly used non-OPs pesticides.

Application of Maillard reaction product of xylose-pea protein enzymatic hydrolysate in 3D printing.

This paper aims to provide a basis for the application of Maillard reaction products and ideas for 3D printing of functional food. We evaluated the effects of pea protein enzymatic hydrolysate-xylose Maillard reaction products (xMRPs) on the printability of 3D printing materials and the structure using various techniques. It was found that compared with the addition of enzymatic hydrolysate, the materials with xMRPs and 3D printing could significantly improve the structure, physical properties, formability of 3D printing materials and 3D print products, among which the xMRPs with 6 g of xylose had the most significant effect. This study has important scientific value and practical significance for the high-valued application of Millard reaction products and the promotion of the practical application of 3D printing technology in the food industry. © 2020 Society of Chemical Industry.

A novel functional C1 inhibitor activity assay in dried blood spot for diagnosis of Hereditary angioedema

BackgroundHereditary angioedema (HAE) is a rare genetic disease caused by deficiency or dysfunction of C1 esterase inhibitor (C1-INH). Timely and accurate diagnosis is an ongoing challenge. Measurement of plasma C1-INH activity is currently the critical standard test. We describe a novel and highly robust point-of-care assay to quantify C1-INH activity in dried blood spot (DBS). MethodsC1-INH was extracted from 3 mm punches of DBS samples and incubated with excess amount of C1 esterase (C1s). The mixture was subsequentially incubated with C1s substrate, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation of the enzyme reaction product. ResultsThe assay was validated within a quantification range from 100 to 1500 mU/mL. The intra-day precision and accuracy ranged from 4.0% to 11.6% and −11.1% to −2.1%, and the inter-day precision and accuracy were 8.1–13.1% and −10.3% to 0.9%, respectively. Normal C1-INH activity (n = 103) ranged from 311 to 1090 mU/mL, whereas 23 out of 24 HAE patients exhibited C1-INH activity lower than 100 mU/mL. ConclusionDBS specimen collection for measurement of functional C1-INH activity in a physician’s office is straightforward and not limited by logistic considerations and therefore, appropriate for the diagnosis of HAE in high throughput diagnostic laboratories.

A Carbamoylase-Based Bioassay for the Detection of Paralytic Shellfish Poisoning Toxins.

Out of control proliferation of toxic phytoplankton, called harmful algal blooms (HABs), have a significant economic impact on bivalve aquaculture and harvesting in coastal waters. Some phytotoxins, such as paralytic shellfish toxins (PSTs), are of concern due to the life-threatening symptoms they can cause. Development of rapid and low-cost screening tools would be a welcome addition to the laboratory methodologies employed in routine monitoring programs. However, most of the assays and biosensors for the screening of PSTs, are restricted to a single target, saxitoxin (STX), which is the most potent PST. The present study aimed at developing an assay for the detection of N-sulfocarbamoyl PST—GTX5, which is one of the most abundant toxins in bivalves during G. catenatum blooms as found on the Portuguese coast. Enzymatic assay employing PSTs’ transforming enzyme—carbamoylase—was proposed. Carbamoylase was extracted and purified from the surf clam S. solida. Carbamoylase displayed similar specificity to both carbamate (STX) and N-sulfocarbamate toxins (GTX5 and C1+2) converting them into decarbamoyl saxitoxin (dcSTX) and decarbamoyl gonyautoxins 2+3 (dcGTX2+3), respectively. The enzymatic assay involved hydrolysis of GTX5 by carbamoylase and quantification of the product of enzymatic reaction, dcSTX, using a potentiometric chemical sensor. A potentiometric sensor with plasticized PVC membrane that displayed sensitivity to dcSTX and selectivity in the presence of GTX5 was employed. Enzymatic assay allowed determination of GTX5 in the concentration range from 0.43 to 3.30 µmolL−1, which encompasses levels of GTX5 in contaminated bivalve extracts with toxicities above PSTs regulatory limits. The feasibility of the carbamoylase-based potentiometric assay for detection of GTX5 was demonstrated.

Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5′‐Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols

AbstractAdenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S‐adenosyl‐l‐methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)‐adenosylhopane, indicating that HpnH catalyzes stereoselective C−C formation between C29 of diploptene and C5′ of 5′‐deoxyadenosine. Further, the HpnH reaction in D2O‐containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C35 bacteriohopane skeleton.

Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5'-Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols.

Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S-adenosyl-l-methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)-adenosylhopane, indicating that HpnH catalyzes stereoselective C-C formation between C29 of diploptene and C5' of 5'-deoxyadenosine. Further, the HpnH reaction in D2 O-containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C35 bacteriohopane skeleton.

Molecular Level Analysis Reveals Changes in Chemical Composition of Dissolved Organic Matter From South Texas Rivers After High Flow Events

Riverine dissolved organic matter (DOM) is a major source of reduced carbon exported from land to marine environments, and the inflow of riverine organic matter greatly affects biogeochemical cycling in estuaries and bays. Thus, any change in DOM composition, such as changes caused by flood waters as a result of storms and hurricanes, can subsequently affect estuarine environments. To investigate the impact of high flow events on riverine DOM, multidimensional molecular level information of DOM from four south Texas Rivers (Aransas, Lavaca, Mission, and Nueces Rivers) was acquired using high-resolution Ion Mobility Quadrupole Time of Flight Liquid Chromatography Mass Spectrometry (IM Q-TOF LCMS). Base-flow samples were collected in May, July and October of 2016, June of 2017, and March of 2018, while high-flow samples were collected in September of 2017, and June and September of 2018. Based on the molecular formula assigned from IM Q-TOF LCMS, H/C ratio decreased during high-flow event (1.52 to 1.51 in ESI+; 1.19 to 1.07 in ESI-), while O/C ratio increased (0.31 to 0.33 in ESI-). Furthermore, DOM shifted from a protein-like and lipid-like dominated community at base flow condition, to a lignin, tannin and condensed aromatic structure dominated one during high flow event based on MS and tandem MS data. These changes in high-flow riverine DOM indicate an increase of terrestrial signal, which likely is a result of mobilization of terrestrial organic matter from the watersheds by flooding. These mobilized DOM, though refractory at high-flow condition in rivers, could be reactive in coastal regions when condition changes, and thus potentially fuel microbial activities downstream. In addition, about 3.76 – 21.8% of DOM molecules contain structural isomers among different flow conditions. This low number of isomer percentages suggests that as the products of various enzymatic biochemical reactions, the number of isomers in DOM is constrained. Taken together, our study provides insights into structural changes of riverine DOM in response to extreme climate events in subtropical regions and have implications in understanding biogeochemical changes in estuaries under a changing climate.

Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01–500 nM) and an estrogen-sensitive yeast bioassay (1–10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50–10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.

Abstract 2151: Monitoring AMP Concentration,the universal product of diverse enzymatic reactions

Abstract Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.

Rat liver folate metabolism can provide an independent functioning of associated metabolic pathways

Folate metabolism in mammalian cells is essential for multiple vital processes, including purine and pyrimidine synthesis, histidine catabolism, methionine recycling, and utilization of formic acid. It remains unknown, however, whether these processes affect each other via folate metabolism or can function independently based on cellular needs. We addressed this question using a quantitative mathematical model of folate metabolism in rat liver cytoplasm. Variation in the rates of metabolic processes associated with folate metabolism (i.e., purine and pyrimidine synthesis, histidine catabolism, and influxes of formate and methionine) in the model revealed that folate metabolism is organized in a striking manner that enables activation or inhibition of each individual process independently of the metabolic fluxes in others. In mechanistic terms, this independence is based on the high activities of a group of enzymes involved in folate metabolism, which efficiently maintain close-to-equilibrium ratios between substrates and products of enzymatic reactions.

A novel beta-1,4 glucanase produced by symbiotic Bacillus sp. CF96 isolated from termite (Anacanthotermes)

A novel beta-1,4-glucanase was purified and characterized from symbiotic Bacillus sp. CF96 of termite. The SDS-PAGE and zymogram analyses revealed a molecular mass of 35.6 kDa. Optimal activity was at 50 °C and pH 5.5, while the enzyme was active over a wide range of temperature 20-80 °C and pH 4-10 and interestingly more than 60% of the maximum activity remained up to pH 9. The enzyme activity increased in the presence of hexane, chloroform and methanol (20% v/v). while, the enzyme activity was inhibited by metal ions such as Mn2+, Hg2+, Cu2+, Zn2+, Mg2+, Fe2+. The isolated enzyme was able to degrade carboxymethyl cellulose (CMC), avicel and cellulose. Cellobiose was the hydrolytic product of enzymatic reaction based on thin layer chromatography (TLC) analysis. Regarding beta-1,4 endo/exoglucanase activity and high temperature, pH and solvent stability, the enzyme has potential for various industrial applications especially in designing pesticide for termite.

Customizing Morphology, Size, and Response Kinetics of Matrix Metalloproteinase-Responsive Nanostructures by Systematic Peptide Design.

Overexpression and activation of matrix metalloproteinase-9 (MMP-9) is associated with multiple diseases and can serve as a stimulus to activate nanomaterials for sensing and controlled release. In order to achieve autonomous therapeutics with improved space-time targeting capabilities, several features need to be considered beyond the introduction of an enzyme-cleavable linker into a nanostructure. We introduce guiding principles for a customizable platform using supramolecular peptide nanostructures with three modular components to achieve tunable kinetics and morphology changes upon MMP-9 exposure. This approach enables (1) fine-tuning of kinetics through introduction of ordered/disordered structures, (2) a 12-fold variation in hydrolysis rates achieved by electrostatic (mis)matching of particle and enzyme charge, and (3) selection of enzymatic reaction products that are either cell-killing nanofibers or disintegrate. These guiding principles, which can be rationalized and involve exchange of just a few amino acids, enable systematic customization of enzyme-responsive peptide nanostructures for general use in performance optimization of enzyme-responsive materials.

Illuminating the catalytic core of ectoine synthase through structural and biochemical analysis

Ectoine synthase (EctC) is the signature enzyme for the production of ectoine, a compatible solute and chemical chaperone widely synthesized by bacteria as a cellular defense against the detrimental effects of osmotic stress. EctC catalyzes the last step in ectoine synthesis through cyclo-condensation of the EctA-formed substrate N-gamma-acetyl-L-2,4-diaminobutyric acid via a water elimination reaction. We have biochemically and structurally characterized the EctC enzyme from the thermo-tolerant bacterium Paenibacillus lautus (Pl). EctC is a member of the cupin superfamily and forms dimers, both in solution and in crystals. We obtained high-resolution crystal structures of the (Pl)EctC protein in forms that contain (i) the catalytically important iron, (ii) iron and the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid, and (iii) iron and the enzyme reaction product ectoine. These crystal structures lay the framework for a proposal for the EctC-mediated water-elimination reaction mechanism. Residues involved in coordinating the metal, the substrate, or the product within the active site of ectoine synthase are highly conserved among a large group of EctC-type proteins. Collectively, the biochemical, mutational, and structural data reported here yielded detailed insight into the structure-function relationship of the (Pl)EctC enzyme and are relevant for a deeper understanding of the ectoine synthase family as a whole.

A non-enzymatic electrochemical biosensor based on Au@PBA(Ni–Fe):MoS2 nanocubes for stable and sensitive detection of hydrogen peroxide released from living cells

Hydrogen peroxide (H2O2) is the main product of enzymatic reactions and plays an important role in biological processes. The detection of H2O2 inside organisms or cells is critical. Here, we report a nickel-iron Prussian blue analogue nanocube doped with molybdenum disulfide and Au nanoparticles (Au@PBA(Ni-Fe):MoS2) as an electrochemical sensing material for the stable detection of H2O2 in neutral solutions for a long time. First, the Prussian blue analogue (PBA(Ni-Fe)) is synthesized by a simple charge-assembly technology, and then etched into PBA(Ni-Fe):MoS2 hollow nanocubes by a high-temperature hydrothermal reaction. Finally, Au nanoparticles are reduced inside the PBA(Ni-Fe):MoS2in situ to generate Au@PBA(Ni-Fe):MoS2 nanocubes. Ni-doping enhances the nanocube's stability in neutral solutions; as a result, the sensor can maintain a stable current response towards H2O2 reduction for more than 1 h. The sensing material can meet the needs of a long-time test. The introduction of Au enhances the electron transfer efficiency, which endows the sensor with good reduction ability for H2O2 at 0 V over a wide linear range (0.5-200 μM and 210-3000 μM) and with a low detection limit (0.23 μM (S/N = 3)), which fulfills the requirements for the detection of H2O2 in a biological system. The sensor can sense H2O2 released from cells stimulated by ascorbic acid. Au@PBA(Ni-Fe):MoS2 provides good guidance for the future development of efficient biosensors to be applied in cell biology.

Highly sensitive multiplex protein detection by droplet‐free digital ELISA

AbstractDigital enzyme‐linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. On the other hand, we have developed a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In this study, we present a method that enables the multiplex detection of proteins based on Droplet‐free Digital ELISA. First, two types of paramagnetic beads immobilized with antibodies specific to different target proteins were incubated with a sample solution, and target proteins were captured on their specific beads. Then, these beads were labeled with horseradish peroxidase (HRP), and tyramide substrate reacted with HRP on beads, resulting in products of this reaction deposited on those beads. This signal amplification on beads makes it possible to count the number of labeled beads digitally. We used this approach to simultaneously detect IL‐6 and HBs Ag with single molecule resolution. The obtained limit of detections were 0.1 pg/mL and 0.013 IU/mL, respectively. Our method has potential applications in simple in vitro diagnostic systems for simultaneous and high‐sensitive detection of protein biomarkers.

Quantification of 11 enzyme activities of lysosomal storage disorders using liquid chromatography-tandem mass spectrometry

AstractLysosomal storage disorders (LSDs) are characterized by the accumulation of lipids, glycolipids, oligosaccharides, mucopolysaccharides, and other biological substances because of the pathogenic deficiency of lysosomal enzymes. Such diseases are rare; thus, a multiplex assay for these disorders is effective for the identification of affected individuals during the presymptomatic period. Previous studies have demonstrated that such assays can be performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection. An assay procedure to quantify the activity of 11 enzymes associated with LSDs was provided. First, a validation study was performed using dried blood spot (DBS) samples with 100% and 5% enzyme activity for quality control (QC). Under the assay condition, the analytical range, defined as the ratio of the peak area of the enzyme reaction products from the DBS for QC with 100% enzyme activity to that from the filter paper blank sample, was between 14 for GALN and 4561 for GLA. Based on these results, the distribution of the enzyme activity for the 11 LSD enzymes was further examined. Consistent with the previous data, the enzyme activity exhibited a bell-shaped distribution with a single peak. The averaged enzyme activity for the healthy neonates was as follows: GLA, 3.80 ± 1.6; GAA, 10.6 ± 4.8; IDUA, 6.4 ± 2.3; ABG, 8.6 ± 3.1; ASM, 3.3 ± 1.1; GALC, 2.8 ± 1.3; ID2S, 16.7 ± 6.1; GALN, 1.2 ± 0.5; ARSB, 17.0 ± 8.7; NAGLU, 4.6 ± 1.5; and GUSB, 46.6 ± 19.0 μmol/h/L (mean ± SD, n = 200). In contrast, the enzyme activity in disease-affected individuals was lower than the minimum enzyme activity in healthy neonates. The results demonstrate that the population of disease-affected individuals was distinguished from that of healthy individuals by the use of LC-MS/MS.