Development of a colorimetric enzymatic assay method for aromatic biogenic monoamine-producing decarboxylases.
Ontology highlight
ABSTRACT: Supplementary information
The online version contains supplementary material available at 10.1007/s10068-021-00938-4.
SUBMITTER: Kim YC
PROVIDER: S-EPMC8302707 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.
| S-EPMC5357303 | biostudies-literature
Project description:Pyruvate decarboxylase (PDC) is a key enzyme in homoethanol fermentation process, which decarboxylates 2-keto acid pyruvate into acetaldehyde and carbon dioxide. PDC enzymes from potential ethanol-producing bacteria such as Zymomonas mobilis, Zymobacter palmae and Sarcina ventriculi have different K(m) and k(cat) values for the substrate pyruvate at their respective optimum pH. In this study, the putative three-dimensional structures of PDC dimer of Z. palmae PDC and S. ventriculi PDC were generated based on the X-ray crystal structures of Z. mobilis PDC, Saccharomyces cerevisiae PDC form-A and Enterobacter cloacae indolepyruvate decarboxylase in order to compare the quaternary structures of these bacterial PDCs with respect to enzyme-substrate interactions, and subunit-subunit interfaces that might be related to the different biochemical characteristics. The PROCHECK scores for both models were within recommended intervals. The generated models are similar to the X-ray crystal structure of Z. mobilis PDC in terms of binding modes of the cofactor, the position of Mg(2+), and the amino acids that form the active sites. However, subunit-subunit interface analysis showed lower H-bonding in both models compared with X-ray crystal structure of Z. mobilis PDC, suggesting a smaller interface area and the possibility of conformational change upon substrate binding in both models. Both models have predicted lower affinity towards branched and aromatic 2-keto acids, which correlated with the molecular volumes of the ligands. The models shed valuable information necessary for further improvement of PDC enzymes for industrial production of ethanol and other products.
| S-EPMC2951667 | biostudies-literature
Project description:Hydrogen sulfide is an important biological signaling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (?max = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ? = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated submicromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications.
| S-EPMC3730526 | biostudies-literature
Project description:Adenosine deaminase (ADA), which catalyzes the irreversible deamination of adenosine to inosine, is related to various human diseases such as tuberculous peritonitis and leukemia. Therefore, the method used to detect ADA activity and screen the effectiveness of various inhibitor candidates has important implications for the diagnosis treatment for various human diseases. A simple and rapid assay method for ADA, based on the enzymatic formation of a luminescent lanthanide complex, is proposed in this study. Inosine, an enzymatic product of ADA with stronger sensitization efficiency for Tb3+ than adenosine, produced a strong luminescence by forming an inosine-Tb3+ complex, and it enabled the direct monitoring of ADA activity in real-time. By introducing only Tb3+ to adenosine and ADA in the buffer, the enhancement of luminescence enabled the detection of a low concentration of ADA (detection limit 1.6 U/L). Moreover, this method could accurately determine the inhibition efficiency (IC50) of the known ADA inhibitor, erhythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and the inhibition of ADA could be confirmed by the naked eye. Considering its simplicity, this assay could be extended to the high-throughput screening of various ADA inhibitor candidates.
| S-EPMC6631010 | biostudies-literature
Project description:Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3 , the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.
| S-EPMC4353337 | biostudies-literature
Project description:Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058-10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).
| S-EPMC4376533 | biostudies-literature
Project description:Worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. Colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). The detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. In this study, we propose a colorimetric assay based on an extended form of double-stranded DNA (dsDNA) self-assembly shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) for detection of Middle East respiratory syndrome coronavirus (MERS-CoV). This platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (LSPR) shift and color changes of AuNPs in the UV-vis wavelength range. We designed a pair of thiol-modified probes at either the 5' end or 3' end to organize complementary base pairs with upstream of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV. The dsDNA of the target and probes forms a disulfide-induced long self-assembled complex, which protects AuNPs from salt-induced aggregation and transition of optical properties. This colorimetric assay could discriminate down to 1 pmol/?L of 30 bp MERS-CoV and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings.
| S-EPMC7119221 | biostudies-literature
Project description:Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO(2) and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO(2) from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO(2) using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O(2) using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate ((13)C(2)-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product (13)CO(2) (m/z 45), avoiding inaccuracies due to endogenous (12)CO(2).
| S-EPMC3164234 | biostudies-literature
Project description:Conjugated polyelectrolytes (CPEs) have been widely used as reporters in colorimetric assays targeting nucleic acids. CPEs provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. Herein, we report a pixelated analysis approach for correlating colorimetric responses of CPE with nucleic acid concentrations down to 1 nM, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. The detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic CPE duplexes and double stranded nucleic acid-CPE triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. Cationic poly[N,N,N-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the CPE reporter deposited on a polyvinylidene fluoride (PVDF) membrane for nucleic acid assay. A smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of CPE on the PVDF membrane, followed by an analysis using the algorithm. The proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, RGB analysis. The obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation.
| S-EPMC7111824 | biostudies-literature
Project description:We report a novel, simple, efficient, and green protocol for biogenic synthesis of silver nanoparticles (AgNPs) in aqueous solution using clove (Syzygium aromaticum) extract as a reducing and protecting agent. Ultraviolet-visible (UV-Vis) spectroscopy was employed to monitor the localized surface plasmon resonance (LSPR) band of clove extract-derived AgNPs prepared under various conditions. Fourier-transform infrared (FTIR) spectroscopy analysis provided information about the surface interaction of the clove extract with the AgNPs. Ultrahigh-resolution transmission electron microscopy (UHRTEM) results confirmed the formation of spherical, uniformly distributed clove extract-capped AgNPs with sizes in the range of 2-20 nm (average size: 14.4 ± 2 nm). Powder X-ray diffractometry analysis (PXRD) illustrated the formation of pure crystalline AgNPs. These AgNPs were tested as a colorimetric sensor to detect trace amounts of vinclozolin (VIN) by UV-Vis spectroscopy for the first time. The AgNP-based sensor demonstrated very sensitive and selective colorimetric detection of VIN, in the range of 2-16 µM (R2 = 0.997). The developed sensor was green, simple, sensitive, selective, economical, and novel, and could detect trace amounts of VIN with limit of detection (LOD) = 21 nM. Importantly, the sensor was successfully employed for the determination of VIN in real water samples collected from various areas in Turkey.
| S-EPMC6915375 | biostudies-literature