Search Results (11)

Blood glutathione (GSH), its oxidized form glutathione disulfide (GSSG), and especially the ratio of reduced to oxidized glutathione (GSH/GSSG) are recognized as robust biomarkers of oxidative stress. However, the broader application of these biomarkers has been limited by two major challenges: (1) the high risk of artifact formation during sample handling, which can artificially increase GSSG levels and bias redox balance measurements, and (2) the reliance on complex, instrument-intensive analytical procedures and the requirement for venous blood. We present an adaptation of the highly sensitive and easy-to-perform Tietze recycling method for microvolumes of blood. The challenge is to achieve accurate and precise measurements while avoiding artifacts, taking advantage of the high sensitivity of this enzymatic recycling analytical procedure. The method uses a simplified sample preparation protocol compatible with small blood volumes (up to 10 μL) and requires only basic laboratory equipment, such as a standard spectrophotometer or microplate reader. As this is an enzyme-based assay, we also carefully evaluate the main factors that can affect the measurements. This novel procedure provides a practical tool for monitoring GSH/GSSG as a biomarker of oxidative stress in various experimental settings by eliminating the need for trained personnel for blood sampling (it is suitable for capillary blood), minimizing discomfort for subjects, and avoiding complex procedures or instruments for analyte detection. Full article

Functional investigations of enzymes involving cellular expression systems are important for pharmacological studies. The precise control of expression is challenging in transiently transfected mammalian cell lines. Here, we explored the ability of Xenopus laevis oocytes to express a membrane-bound enzyme for functional characterization using standard 96-well plates and a fluorescence-based plate reader assay. We microinjected oocytes with cRNA encoding the angiotensin converting enzyme 2 (ACE2) and measured the enzymatic activity in single oocytes using a commercial fluorescence-based assay. The injected oocytes showed up to a 50-fold increase in fluorescence compared to uninjected oocytes. This fluorescence intensity was dose-dependent on the amount of ACE2 cRNA. These results suggest that Xenopus oocytes can be used for the functional evaluation of membrane-bound enzymes, decreasing the experimental workload. Full article

A 96-well microplate reader for absorption spectroscopy was designed, constructed, and tested at a total cost of ca. 500 USD. The reduced cost of the device represents the major technical contribution of this manuscript, as costs were reduced 7 fold from previous reports. The device was able to achieve 3σ limits of detection of ca. 0.01 absorbance units (AU) over a 60 second measurement for the mid-visible wavelength range. Component parts are either commercially available, or 3D printed from plans. Analysis wavelength can be altered throughout the visible region through use of various photographic or theatrical filters. This feature allows the well plate reader to be used for typical laboratory assays such as cell population estimation by optical density (OD) at 600 nm, or enzyme-linked immunosorbent assays (ELISA) at 450 nm. This manuscript reports on the motivation and process of constructing the device, lists required parts, presents data demonstrating device function, and provides the community of scholars with plans to reproduce the work. The device can be reproduced in laboratories lacking sufficient resources to purchase commercially available options and this outcome contributes towards empowerment of individuals and equity of scientific enquiry. Full article

Periodontitis (P) is a highly prevalent inflammatory disease of the oral cavity. The objective of the study was to evaluate the stages of pro-inflammatory cytokine IL-1β in initial, moderate and severe periodontitis. One hundred and twenty two patients were included in the study. Periodontitis subjects had at least 20 natural teeth and ≥8 sites with pocket depths of >4 mm and clinical attachment loss (CAL). A questionnaire was used with respect to the socio demographic parameters which included age, gender, ethnicity, education, marital, residence and occupation. To categorize the severity of the disease, teeth were assessed for, Plaque index (PI), Bleeding on probing (BOP), CAL, missing tooth, tooth mobility and bone loss. Unstimulated whole saliva (UWS) was collected and Interleukin-1β (IL-1β) cytokine levels were analyzed using enzyme linked immunosorbent assay with microplate reader at 450 nm. Clinical parameters and salivary cytokine concentrations were assessed using one-way analysis of variance, whereas a correlation of cases with gender and severity of periodontitis was evaluated using chi-square test. Fifty-nine patients were healthy controls and 63 were periodontitis patients Thirty two percent (n = 20) had initial periodontitis, 40% (n = 25) suffered from moderate and 29% (n = 18) had severe periodontitis. Periodontitis subgroups were significantly different with regards to age and gender (p < 0.001). The mean PPD and CAL among the periodontitis patients (PPD, 3.52 ± 1.25 mm; CAL, 4.04 ± 1.64 mm) were significantly compromised (p < 0.05) compared to healthy controls (PPD, 1.52 ± 0.73 mm; CAL, 0.08 ± 0.28 mm). Increased levels of IL-1β were associated with high CAL and PPD findings. UWS IL-1β levels were higher in periodontitis patients compared to healthy individuals. In addition, cases of severe periodontitis showed significantly higher UWS IL-1β levels compared to initial and moderate periodontitis patients. Comparative levels of salivary IL-1β can be potentially used as a diagnostic tool for periodontitis identification and disease progression along with clinical parameters. Full article

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis–Menten model and is inactivated by H₂O₂. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H₂O₂] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H₂O₂ decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H₂O₂ decomposition by catalase in 32 samples in triplicates if kept below 8 mM min⁻¹ in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost. Full article

Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology. Full article

Polymerase chain reaction (PCR)-based antigen tests are technically difficult, time-consuming, and expensive, and may produce false negative results requiring follow-up confirmation with computed tomography. The global coronavirus disease 2019 (COVID-19) pandemic has increased the demand for accurate, easy-to-use, rapid, and cost-effective antigen tests for clinical application. We propose a de novo antigen test for diagnosing COVID-19 using the combination of sandwich enzyme-linked immunosorbent assay and thio-nicotinamide adenine dinucleotide (thio-NAD) cycling. Our test takes advantage of the spike proteins specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The limit of detection of our test was 2.3 × 10⁻¹⁸ moles/assay. If the virus has ~25 spike proteins on its surface, our method should detect on the order of 10⁻²⁰ moles of virus/assay, corresponding to ~10⁴ copies of the virus RNA/assay. The detection sensitivity approaches that of PCR-based assays because the average virus RNA load used for PCR-based assays is ~10⁵ copies per oro- or naso-pharyngeal swab specimen. To our knowledge, this is the first ultrasensitive antigen test for SARS-CoV-2 spike proteins that can be performed with an easy-to-use microplate reader. Sufficient sensitivity can be achieved within 10 min of thio-NAD cycling. Our antigen test allows for rapid, cost-effective, specific, ultrasensitive, and simultaneous multiple measurements of SARS-CoV-2, and has broad application for the diagnosis for COVID-19. Full article

Advanced oxidation processes represent a viable alternative in water reclamation for potable reuse. Sensing methods of hydrogen peroxide are, therefore, needed to test both process progress and final quality of the produced water. Several bio-based assays have been developed so far, mainly relying on peroxidase enzymes, which have the advantage of being fast, efficient, reusable, and environmentally safe. However, their production/purification and, most of all, batch-to-batch consistency may inherently prevent their standardization. Here, we provide evidence that a synthetic de novo miniaturized designed heme-enzyme, namely Mimochrome VI*a, can be proficiently used in hydrogen peroxide assays. Furthermore, a fast and automated assay has been developed by using a lab-bench microplate reader. Under the best working conditions, the assay showed a linear response in the 10.0–120 μM range, together with a second linearity range between 120 and 500 μM for higher hydrogen peroxide concentrations. The detection limit was 4.6 μM and quantitation limits for the two datasets were 15.5 and 186 μM, respectively. In perspective, Mimochrome VI*a could be used as an active biological sensing unit in different sensor configurations. Full article

Plant-derived phenolic compounds contribute to the defense against various pathogens, including root-lesion nematodes (Pratylenchus spp.). However, there are no reports on the role of phenolic compounds in wheat (Triticum aestivum) against Pratylenchus thornei. In this study, wheat genotypes ranging from resistant to very susceptible to P. thornei were used to investigate the level of total phenols and phenol oxidases, polyphenol oxidase (PPO), and peroxidase (POD) expressed in root tissues when grown in the presence and absence of P. thornei over time (2–8 weeks). Higher constitutive levels of total phenols were found in resistant synthetic hexaploid wheats CPI133872 (576 µg gallic acid equivalent (GAE)/g root) and CPI133859 (518 µg GAE/g root) at 8 weeks after sowing, compared with moderately resistant and susceptible genotypes (192 to 390 µg GAE/g root). The activity of PPO was induced in resistant (CPI133872) and moderately resistant (GS50a and its derivate QT8343) genotypes, becoming maximal at 4 weeks after P. thornei inoculation. The activity of POD was induced in CPI133872 at 6 weeks after P. thornei inoculation. Different genetic sources of resistance to P. thornei showed diverse defense mechanisms and differences in timing responses. The combined effects of total phenols and oxidative enzymes could be important for defense against P. thornei in some resistant wheat genotypes. Full article

The aim of the study was to examine antibacterial properties of microemulsion structure produced from Aloe vera var. littoralis extract as a new tool of nanoscale drug-like materials. Aloe vera var. littoralis (A. littoralis) extract was prepared by distillation method. A nonocarrier structure in the microemulsion system was prepared from the extract. Serial concentrations were prepared from 8 mg/mL extract and the nonocarrier containing 0.1 mg/mL pure extract and were evaluated by a disk diffusion method for 35 Salmonella clinical isolates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by microbroth dilution assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method by an enzyme-linked immunosorbent assay(ELISA) Microplate Reader apparatus. Antioxidant activity of the extract was determined by measuring the ferric reducing ability of plasma (FRAP) assay. From 35 clinical isolates of Salmonella, 17 isolates—including resistant isolates of S.E.1103 and S.E.49—had a zone of inhibition (ZI) of 7 to 32 mm in 0.007 mg/mL of the extract. S.E.76 isolate exposed to 30 µg/mL ceftazidime disk had a ZI of 12 mm but had 10 mm in 7µg/mL of A. littoralis extract. The inhibitory effect of a nanocarrier at a concentration of 25 µg/mL by 20 mm ZI was comparable by the ceftazidime (30 µg/mL) effect. MIC₅₀ was 0.25 mg/mL and MBC₅₀ was 0.5 mg/mL by MTT method for the extract. It was shown that A.littoralis extract had antioxidant activity of 31.67 µM/mg that could be increased based on concentration. It was concluded that the nanocarrier had a significant effect on the studied isolates in comparison with ordinary antibiotics and had potential for use as a natural antioxidant and antimicrobial material in complementary medicine. Full article

The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings. Full article