scholarly journals A Fluorescence Kinetic-Based Aptasensor Employing Stilbene Isomerization for Detection of Thrombin

Materials ◽  
2021 ◽  
Vol 14 (22) ◽  
pp. 6927
Author(s):  
Xinling Zeng ◽  
Qing Zhou ◽  
Liyan Wang ◽  
Xiaoxian Zhu ◽  
Kuiyan Cui ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Disodium Salt ◽  
Salt Hydrate ◽  
Satisfactory Performance ◽  
Attenuation Rate ◽  
Detection Mode ◽  
One Step ◽  
Analyte Detection ◽  
Analytical Approaches ◽  
Fluorescence Kinetic

It is important to detect thrombin due to its physiological and pathological roles, where rapid and simple analytical approaches are needed. In this study, an aptasensor based on fluorescence attenuation kinetics for the detection of thrombin is presented, which incorporates the features of stilbene and aptamer. We designed and synthesized an aptasensor by one-step coupling of stilbene compound and aptamer, which employed the adaptive binding of the aptamer with thrombin to cause a change in stilbene fluorescence attenuation kinetics. The sensor realized detection of thrombin by monitoring the variation in apparent fluorescence attenuation rate constant (kapp), which could be further used for probing of enzyme–aptamer binding. In comprehensive studies, the developed aptasensor presented satisfactory performance on repeatability, specificity, and regeneration capacity, which realized rapid sensing (10 s) with a limit of detection (LOD) of 0.205 μM. The strategy was successful across seven variants of thrombin aptasensors, with tunable kapp depending on the SITS (4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate) grafting site. Analyte detection mode was demonstrated in diluted serum, requiring no separation or washing steps. The new sensing mode for thrombin detection paves a way for high-throughput kinetic-based sensors for exploiting aptamers targeted at clinically relevant proteins.

scholarly journals Highly sensitive dual mode electrochemical platform for microRNA detection

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Pawan Jolly ◽  
Marina R. Batistuti ◽  
Anna Miodek ◽  
Pavel Zhurauski ◽  
Marcelo Mulato ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Dual Mode ◽  
Electrode Surfaces ◽  
Microrna Detection ◽  
Highly Sensitive ◽  
Detection Mode ◽  
Amplification Strategy

Abstract MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

scholarly journals Overview of Portable Assays for the Detection of Mycotoxins, Allergens, and Sanitation Monitoring

2020 ◽  
Author(s):  
Ronald W Sarver ◽  
David J Almy ◽  
Eric R Bergeron ◽  
Benjamin F Strong ◽  
Brent A Steiner ◽  
...  
Keyword(s):  
Disodium Salt ◽  
Distillers Grains ◽  
Diagnostic Assay ◽  
Salt Hydrate ◽  
Protein Assay ◽  
Diagnostic Assays ◽  
Diagnostic Kits ◽  
Environmental Surfaces ◽  
Food Recalls ◽  
Sources Of Contamination

Abstract Background Many food recalls are related to the presence of undeclared allergens and microorganisms in food products. To reduce these occurrences, portable diagnostic assay kits are available to quantitate mycotoxins, to detect allergens and gluten in foods and on environmental surfaces, and for sanitation monitoring. Objective This article reviews diagnostic kits that can detect sources of contamination in food and ingredients as well as on surfaces and clean-in-place rinses. Method Mycotoxins and gluten were detected using lateral flow diagnostic assays. Sanitation monitoring of surfaces was completed using a chemiluminescent assay to detect adenosine 5′-triphosphate disodium salt hydrate (ATP) and another assay to detect protein. Results Gluten was detected at 10 ppm in spiked commodities and on wet and dry surfaces at 2.5 µg/100cm2. Deoxynivalenol was quantitated in dry distillers grains plus solubles and mean results were within two SDs of those determined by HPLC. The chemiluminescent assay had an LOD of 6 fmol of ATP and was able to detect a 1:10 000 dilution of orange juice from surfaces. The protein assay detected 5 µg of bovine serum albumin (BSA) directly applied to the sampler, 100 µg of BSA on surfaces, and detected 1:10 dilutions of Greek yogurt and raw beef from surfaces. Conclusions Portable diagnostic kits evaluated in this work provided accurate, rapid, and sensitive results for detection of mycotoxins, gluten, proteins, and ATP. These methods can be used in facilities with minimal training and provide results that are important to ensure food safety. Highlights Portable methods to detect gluten, mycotoxins, proteins, and ATP are presented.

scholarly journals Canine distemper virus detection by different methods of One-Step RT-qPCR

2016 ◽  
Vol 46 (9) ◽  
pp. 1601-1606
Author(s):  
Claudia de Camargo Tozato ◽  
Vívian Ferreira Zadra ◽  
Caroline Rodrigues Basso ◽  
João Pessoa Araújo Junior
Keyword(s):  
Urine Sample ◽  
Canine Distemper Virus ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Canine Distemper ◽  
System A ◽  
Virus Rna ◽  
One Step ◽  
Commercial Kits ◽  
The One

ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

High Throughput Determination of BTEX by a One-Step Fluorescence Polarization Immunoassay

2005 ◽  
Vol 2 (3) ◽  
pp. 227 ◽  
Author(s):  
Sergei A. Eremin ◽  
Dietmar Knopp ◽  
Reinhard Niessner ◽  
Ji Youn Hong ◽  
Song-Ja Park ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Petroleum Products ◽  
Cross Reactivity ◽  
Rapid Screening ◽  
Fluorescence Polarization Immunoassay ◽  
Phenylcarboxylic Acids ◽  
One Step ◽  
Benzene Toluene

Environmental Context.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are used as solvents in paints and coatings and are constituents of petroleum products. BTEX can contaminate air, water or soil and is toxic; benzene, in particular, is a recognized human carcinogen. Most existing methods for detecting BTEX are time-consuming, complicated and very expensive for routine screening. A rapid immunoassay of BTEX is presented that greatly simplifies environmental monitoring of water contamination. Abstract.For the rapid screening of BTEX (benzene, toluene, ethylbenzene, xylenes), a fluorescence polarization immunoassay (FPIA) was developed using the fluorescence polarization analyzer Abbott TDx. Several fluorescence-labelled tracers were synthesized by binding ethylenediamine fluorescein thiocarbamyl (EDF) to various substituted phenylcarboxylic acids. The binding of 27 tracers with two antisera that can be considered as class-specific for BTEX was investigated to select optimal tracer–antibody pairs. Significant differences were found in binding, titer, sensitivity, and assay kinetics. A best pair of anti-BTEX antiserum and EDF-labelled p-tolylacetic acid tracer was selected for FPIA. To simplify the method, an immunocomplex single reagent was prepared to detect BTEX by a one-step FPIA. One-step FPIA is a rapid homogeneous type of immunoassay that has only one pipetting step, does not need separation of free and bound analyte and can be performed at room temperature. The within-run coefficient of variation was ranged between 3.4% and 5.7%. Furthermore, if the measurement can be done at constant temperature, standards for every assay run are unnecessary. Cross-reactivity studies of petroleum compounds and a BTEX mixture indicated that p-xylene was most reactive with a limit of detection (LOD) of 0.22 µg mL−1 in 50 µL of sample. The LOD for toluene and benzene was 2.1 and 11 µg mL−1 respectively. The immunocomplex single reagent has proven to be significantly more stable than the corresponding solutions of antibody and tracer.

scholarly journals Detection of Melamine Based on the Fluorescence Changes of Nitrogen-Doped Carbon Dots

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Wenzhi Yin ◽  
Chaoqun Ma ◽  
Tuo Zhu ◽  
Jiao Gu ◽  
Chun Zhu ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Detection Method ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Mercury Ions ◽  
Measuring Range ◽  
Nitrogen Doped ◽  
One Step ◽  
Doped Carbon Dots ◽  
Nitrogen Doped Carbon

In order to determine the concentration of melamine, nitrogen-doped carbon dots (NCDs) were synthesized in one step as a fluorescent probe. Uric acid and diethylenetriamine were used as carbon source and nitrogen source, respectively. The experimental results showed that the fluorescence of NCDs can be quenched by mercury ions (Hg2+). Due to the strong coordination affinity between the carbon-nitrogen heterocyclic of melamine and Hg2+, part of Hg2+ coordinated with melamine when melamine was mixed with Hg2+. Then, the fluorescence of the added NCDs was quenched by the remaining Hg2+. Therefore, the concentration of melamine could be determined. The results show that the method has high sensitivity in wide measuring range that the linear ranges are 50–400 μg/L and 800–2500 μg/L, and the R2 is 0.997 and 0.988, respectively, with the limit of detection (LOD) of 21.76 μg/L. The NCDs are easy to fabricate, and the detection method is easy to implement. In this study, a new method for melamine detection was established, and the proposed method for melamine detection can provide some insights for food safety detection.

scholarly journals One-Step Preparation of Nitrogen-Doped Graphene Quantum Dots With Anodic Electrochemiluminescence for Sensitive Detection of Hydrogen Peroxide and Glucose

2021 ◽  
Vol 9 ◽  
Author(s):  
Zheng Yanyan ◽  
Jing Lin ◽  
Liuhong Xie ◽  
Hongliang Tang ◽  
Kailong Wang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Quantum Dots ◽  
Limit Of Detection ◽  
Hydrothermal Process ◽  
Graphene Quantum Dots ◽  
Sensitive Detection ◽  
Uniform Size ◽  
Nitrogen Doped ◽  
Nitrogen Doped Graphene ◽  
One Step

Simple and efficient synthesis of graphene quantum dots (GQDs) with anodic electrochemiluminescence (ECL) remains a great challenge. Herein, we present an anodic ECL-sensing platform based on nitrogen-doped GQDs (N-GQDs), which enables sensitive detection of hydrogen peroxide (H2O2) and glucose. N-GQDs are easily prepared using one-step molecular fusion between carbon precursor and a dopant in an alkaline hydrothermal process. The synthesis is simple, green, and has high production yield. The as-prepared N-GQDs exhibit a single graphene-layered structure, uniform size, and good crystalline. In the presence of H2O2, N-GQDs possess high anodic ECL activity owing to the functional hydrazide groups. With N-GQDs being ECL probes, sensitive detection of H2O2 in the range of 0.3–100.0 μM with a limit of detection or LOD of 63 nM is achieved. As the oxidation of glucose catalyzed by glucose oxidase (GOx) produces H2O2, sensitive detection of glucose is also realized in the range of 0.7–90.0 μM (LOD of 96 nM).

scholarly journals 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

2021 ◽  
Author(s):  
Gulay Korukluoglu ◽  
Mustafa Kolukirik ◽  
Fatma Bayrakdar ◽  
Gozde Girgin Ozgumus ◽  
Ayse Basak Altas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Clinical Samples ◽  
One Step ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

DEVELOPMENT AND VALIDATION OF RAPID AND SIMPLE BIOANALYTICAL METHOD FOR DOCETAXEL WITH ONE STEP PROTEIN PRECIPITATION

INDIAN DRUGS ◽  
2019 ◽  
Vol 56 (07) ◽  
pp. 23-32
Author(s):  
P. C. Mehendale ◽  
R. B Athawale ◽  
K. K. Singh ◽  
Keyword(s):  
Extraction Efficiency ◽  
Regression Coefficient ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Protein Precipitation ◽  
Isocratic Elution ◽  
Limit Of Quantification ◽  
Extraction Solvent ◽  
One Step ◽  
Development And Validation

A rapid and simple bio-analytical method with one step protein precipitation and extraction using acetonitrile as extraction solvent was developed for docetaxel. The extraction efficiency was 87.81% with satisfactory separation of docetaxel and IS peaks by isocratic elution with C18 column (25 cm X 4.5 mm, 0.5μm), acetonitrile and water (53:47 % V/V) as a mobile phase at ambient temperature and flow rate of 1mL/min. Paclitaxel solution in acetonitrile (10 mcg/ mL) was used as internal standard. The calibration curve was linear over the concentration range 50 – 5000 ng/mL, regression coefficient R2= 0.99936 and slope 0.00034. The limit of quantification and limit of detection were found to be 33 ng/ mL and 100 ng/mL, respectively. Coefficient of variation for within day and between the days was in the range of 10.9 to 14.9 and 12.5 to 15.05, respectively. Accuracy of the method indicated % recovery of 97.92 – 104.24%. Thus, a precise, accurate and robust method was developed and validated as per FDA guidelines.

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