scholarly journals Early Detection of the Fungal Banana Black Sigatoka Pathogen Pseudocercospora fijiensis by an SPR Immunosensor Method

Sensors ◽  
2019 ◽  
Vol 19 (3) ◽  
pp. 465 ◽  
Author(s):  
Donato Luna-Moreno ◽  
Araceli Sánchez-Álvarez ◽  
Ignacio Islas-Flores ◽  
Blondy Canto-Canche ◽  
Mildred Carrillo-Pech ◽  
...  
Keyword(s):  
Matrix Effects ◽  
Limit Of Detection ◽  
Black Sigatoka ◽  
Leaf Extracts ◽  
Self Assembled Monolayer ◽  
Early Stages ◽  
Detection And Identification ◽  
Response Range ◽  
Spr Immunosensor ◽  
Pseudocercospora Fijiensis

Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL−1, a sensitivity of 0.0021 units of reflectance per ng mL−1 and a linear response range for the antigen from 39.1 to 122 µg mL−1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.

scholarly journals Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 10
Author(s):  
Ilenia Drigo ◽  
Elena Tonon ◽  
Simone Pascoletti ◽  
Fabrizio Anniballi ◽  
Suzanne R. Kalb ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Animal Health ◽  
Lethal Dose ◽  
Reference Method ◽  
Mouse Bioassay ◽  
Peptide Fragments ◽  
Mass Spectrometers ◽  
Detection And Identification ◽  
Maldi Biotyper ◽  
Sensitivity Specificity

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66–100%), 96.08% (95% CI: 86.54–99.52%), and 93.33% (95% CI: 78.25–98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15–99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

scholarly journals Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 180
Author(s):  
Lucia Sarcina ◽  
Giuseppe Felice Mangiatordi ◽  
Fabrizio Torricelli ◽  
Paolo Bollella ◽  
Zahra Gounani ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Covalent Binding ◽  
Hiv Infections ◽  
Selectivity Ratio ◽  
Selective Detection ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Label Free Detection ◽  
P24 Protein ◽  
Hiv 1

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

scholarly journals Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection

Chemosensors ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 49
Author(s):  
Pushap Raj ◽  
Man Hwan Oh ◽  
Kyudong Han ◽  
Tae Yoon Lee
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Cost Effective ◽  
Covalent Immobilization ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Detection Range ◽  
Linear Detection

Bacterial infections have become a significant challenge in terms of public health, the food industry, and the environment. Therefore, it is necessary to address these challenges by developing a rapid, cost-effective, and easy-to-use biosensor for early diagnosis of bacterial pathogens. Herein, we developed a simple, label-free, and highly sensitive immunosensor based on electrochemical detection using the Au@MoS₂–PANI nanocomposite. The conductivity of the glassy carbon electrode is greatly enhanced using the Au@MoS₂–PANI nanocomposite and a self-assembled monolayer of mercaptopropionic acid on the gold nanoparticle surface was employed for the covalent immobilization of antibodies to minimize the nonspecific adsorption of bacterial pathogens on the electrode surface. The biosensor established a high selectivity and sensitivity with a low limit of detection of 10 CFU/mL, and detected Escherichia coli within 30 min. Moreover, the developed biosensor demonstrated a good linear detection range, practical utility in urine samples, and electrode regenerative studies.

scholarly journals Validation of a New Sensitive Method for the Detection and Quantification of R and S-Epimers of Ergot Alkaloids in Canadian Spring Wheat Utilizing Deuterated Lysergic Acid Diethylamide as an Internal Standard

Toxins ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 22
Author(s):  
Jensen Cherewyk ◽  
Taylor Grusie-Ogilvie ◽  
Barry Blakley ◽  
Ahmad Al-Dissi
Keyword(s):  
Spring Wheat ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Ergot Alkaloids ◽  
Internal Standard ◽  
Lysergic Acid ◽  
Lysergic Acid Diethylamide ◽  
Relative Standard ◽  
Validation Parameters ◽  
Sensitive Method

Ergot sclerotia effect cereal crops intended for consumption. Ergot alkaloids within ergot sclerotia are assessed to ensure contamination is below safety standards established for human and animal health. Ergot alkaloids exist in two configurations, the R and S-epimers. It is important to quantify both configurations. The objective of this study was to validate a new ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantification of six R and six S-epimers of ergot alkaloids in hard red spring wheat utilizing deuterated lysergic acid diethylamide (LSD-D3) as an internal standard. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effects, recovery and precision were investigated. For the 12 epimers analyzed, low LOD and LOQ values were observed, allowing for the sensitive detection of ergot epimers. Matrix effects ranged between 101–113% in a representative wheat matrix. Recovery was 68.3–119.1% with an inter-day precision of <24% relative standard deviation (RSD). The validation parameters conform with previous studies and exhibit differences between the R and S-epimers which has been rarely documented. This new sensitive method allows for the use of a new internal standard and can be incorporated and applied to research or diagnostic laboratories.

Assessing matrix, interferences and comparability between the Abbott Diagnostics and the Beckman Coulter high-sensitivity cardiac troponin I assays

2018 ◽  
Vol 56 (7) ◽  
pp. 1176-1181 ◽  
Author(s):  
Peter A. Kavsak ◽  
Paul Malinowski ◽  
Chantele Roy ◽  
Lorna Clark ◽  
Shana Lamers
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Mean Difference ◽  
Plasma Samples ◽  
Matrix Interferences ◽  
Heparin Plasma ◽  
Edta Plasma

Abstract Background: Analytical evaluation of high-sensitivity cardiac troponin (hs-cTn) assays, with particular attention to imprecision, interferences and matrix effects, at normal cTn concentrations, is of utmost importance as many different clinical algorithms use concentration cutoffs <10 ng/L for decision-making. The objective for the present analytical study was to compare the new Beckman Coulter hs-cTnI assay (Access hsTnI) to Abbott’s hs-cTnI assay in different matrices and for different interferences, with a focus on concentrations <10 ng/L. Methods: The limit of blank (LoB) and the limit of detection (LoD) were determined in different matrices for the Beckman hs-cTnI assay. Passing-Bablok regression and difference plots were determined for 200 matched lithium heparin and EDTA plasma samples for the Beckman assay and 200 lithium heparin samples for the Abbott assay. Both EDTA and heparin plasma samples were also evaluated for stability under refrigerated conditions, for endogenous alkaline phosphatase interference and for hemolysis and icterus. Results: The Beckman hs-cTnI assay LoB was 0.5 ng/L with the following range of LoDs=0.8–1.2 ng/L, with EDTA plasma yielding lower concentrations as compared to lithium heparin plasma (mean difference=−14.9%; 95% CI=−16.9 to 12.9). Below 10 ng/L, lithium heparin cTnI results from the Beckman assay were on average 1.1 ng/L (95% CI=0.7 to 1.5) higher than the Abbott results, with no difference between the methods when using EDTA plasma (mean difference =−0.1 ng/L; 95% CI=−0.3 to 0.2). Low cTnI concentrations were less effected by interferences in EDTA plasma. Conclusions: The Access hsTnI method can reliably detect normal cTnI concentrations with both lithium heparin and EDTA plasma being suitable matrices.

scholarly journals D-shaped plastic optical fibre aptasensor for fast thrombin detection in nanomolar range

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nunzio Cennamo ◽  
Laura Pasquardini ◽  
Francesco Arcadio ◽  
Lia E. Vanzetti ◽  
Alessandra Maria Bossi ◽  
...  
Keyword(s):  
Optical Fibre ◽  
Photoelectron Spectroscopy ◽  
Limit Of Detection ◽  
Low Cost ◽  
Gold Surface ◽  
Coagulation Cascade ◽  
Self Assembled Monolayer ◽  
Detection Range ◽  
Clinical Biomarkers ◽  
Static Contact

AbstractThe development of optical biosensors for the rapid and costless determination of clinical biomarkers is of paramount importance in medicine. Here we report a fast and low-cost biosensor based on a plasmonic D-shaped plastic optical fibre (POF) sensor derivatized with an aptamer specific for the recognition of thrombin, the target marker of blood homeostasis and coagulation cascade. In particular, we designed a functional interface based on a Self Assembled Monolayer (SAM) composed of short Poly Ethylene Glycol (PEG) chains and biotin-modified PEG thiol in ratio 8:2 mol:mol, these latter serving as baits for the binding of the aptamer through streptavidin-chemistry. The SAM was studied by X-ray Photoelectron Spectroscopy (XPS) analysis, static contact angle (CA), Surface Plasmon Resonance (SPR) in POFs, and fluorescence microscopy on gold surface. The optimized SAM composition enabled the immobilization of about 112 ng/cm2 of aptamer. The thrombin detection exploiting POF-Aptasensor occurred in short times (5–10 minutes), the reached Limit of Detection (LOD) was about 1 nM, and the detection range was 1.6–60 nM, indicating the POF-Aptasensor well addresses the needs for a low-cost, simple to use and to realize, rapid, small size and portable diagnostic platform.

scholarly journals Revisiting the Neuroblastoma Cell-Based Assay (CBA-N2a) for the Improved Detection of Marine Toxins Active on Voltage Gated Sodium Channels (VGSCs)

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 281 ◽  
Author(s):  
Jérôme Viallon ◽  
Mireille Chinain ◽  
Hélène Taiana Darius
Keyword(s):  
High Throughput Screening ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Neuroblastoma Cell ◽  
Consensus Protocol ◽  
Limit Of Quantification ◽  
Voltage Gated ◽  
Starting Point ◽  
Fish Samples ◽  
Seafood Products

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

scholarly journals First Draft Genome Sequence Resource of a Strain of Pseudocercospora fijiensis Isolated in North America

2020 ◽  
Vol 110 (10) ◽  
pp. 1620-1622
Author(s):  
Luis Amarillas ◽  
Mitzi Estrada-Acosta ◽  
Rubén G. León-Chan ◽  
Carlos López-Orona ◽  
Luis Lightbourn
Keyword(s):  
North America ◽  
Fungicide Resistance ◽  
Fungal Pathogens ◽  
Genomic Sequence ◽  
Draft Genome ◽  
Bioinformatic Analysis ◽  
Black Sigatoka ◽  
Efficient Management ◽  
Black Sigatoka Disease ◽  
Pseudocercospora Fijiensis

Black Sigatoka disease, caused by the fungus Pseudocercospora fijiensis, is one of the most devastating diseases of banana around the world. Fungicide applications are the primary tool used to manage black Sigatoka, but fungicide resistance in P. fijiensis, as in other fungal pathogens, is one of the major limitations in the efficient management and prevention of this disease. In the current study, we present the draft genome of P. fijiensis strain IIL-20, the first genomic sequence published from a strain of this fungus isolated in North America. Bioinformatic analysis showed putative genes involved in fungus virulence and fungicide resistance. These findings may lead us to a better understanding of the molecular pathogenesis of this fungal pathogen and also to the discovery of the mechanisms conferring fungicide resistance.

Determination of ketamine and norketamine in hair and evaluation of polydrug use in ketamine abusers using hair analysis in Korea

2020 ◽  
Author(s):  
Jongsook Rhee ◽  
Jihyun Kim ◽  
Moonhee Jang ◽  
Ilchung Shi ◽  
Sangki Lee
Keyword(s):  
Mass Spectrometry ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Gas Chromatography Mass Spectrometry ◽  
Controlled Drugs ◽  
Hair Samples ◽  
Limit Of Quantitation ◽  
Liquid Chromatography Tandem Mass ◽  
Forensic Service

Abstract This study evaluated hair samples from 28 subjects who had measurable ketamine levels among the samples requested from 2016 to 2017 into Seoul Institute National Forensic Service in Korea. Ketamine in the hair was extracted by using a solution of 1% hydrochloric acid in methanol for 16 h. Extracts were analyzed using gas chromatography mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS-MS). LC-MS-MS method was validated by determining the limit of detection (LOD), limit of quantitation (LOQ), linearity, intra- and inter-accuracy, precision, and matrix effects. In 59 ketamine-positive hair or hair segments from 28 ketamine abusers, the ketamine concentration was found to be in the range of 0.011-335.8 ng/mg (mean, 13.6; median, 1.8), and the norketamine concentration was found to be in the range of 0.001-35.7 ng/mg (mean, 7.5; median, 0.44). The ratio of norketamine to ketamine concentration in hair was in the range of 0.01-1.46 (mean, 0.34; median, 0.26). The distribution of ketamine concentration in hair samples was as follows: 0.01-0.1 ng/mg in 11 samples (18.6%), 0.1-5 ng/mg in 33 samples (55.9%), 5-10 ng/mg in 4 samples (6.8%), 10-15 ng/mg in 2 samples (3.4%), 15-20 ng/mg in 4 samples (6.8%), 40-45 ng/mg in 2 samples (3.4%), 45-50 ng/mg in 1 samples 1.7%) and &gt;100 ng/mg in only 2 samples (3.4%). In the hair of ketamine-abusers, 26 of 28 subjects had simultaneously ketamine with detectable levels of other controlled drugs, including MDMA (n=9), MA (n=3), MDMA/MA (n=3), MDMA/PMA (n=3), MDMA/PMA/MA (n=2), cocaine (n=1), and other drugs (n=5, propofol, zolpidem or benzodiazepines). In most of the hair samples were detected ketamine with other controlled drugs: MDMA (60.7%), MA (28.6%), PMA(17.9%), zolpidem (17.9%), and propofol (14.3%) in the frequency of abuse. In conclusion, most of the ketamine-abusers (92.9%) would be polydrug abusers, who were concomitantly abusing other controlled substances.

Identification of Suvorexant in Blood Using LC–MS-MS: Important Considerations for Matrix Effects and Quantitative Interferences in Targeted Assays

2019 ◽  
Vol 44 (3) ◽  
pp. 245-255 ◽  
Author(s):  
Britni Skillman ◽  
Sarah Kerrigan
Keyword(s):  
Matrix Effects ◽  
Limit Of Detection ◽  
Chemical Properties ◽  
Internal Standard ◽  
Forensic Toxicology ◽  
Calibration Model ◽  
Physico Chemical ◽  
Limit Of Quantitation ◽  
Liquid Chromatography Tandem Mass ◽  
Electrospray Interface

Abstract Suvorexant (Belsomra®) is a novel dual orexin receptor antagonist used for the treatment of insomnia. The prevalence of suvorexant in forensic samples is relatively unknown, which demonstrates the need for robust analytical assays for the detection of this sedative hypnotic in forensic toxicology laboratories. In this study, suvorexant was isolated from whole blood using a simple acidic/neutral liquid–liquid extraction followed by analysis by liquid chromatography tandem mass spectrometry (LC–MS/MS). Matrix effects were evaluated qualitatively and quantitatively using various extraction solvents, proprietary lipid clean-up devices and source conditions. The method was validated in terms of limit of detection, limit of quantitation, precision, bias, calibration model, carryover, matrix effects and drug interferences. Electrospray is a competitive ionization process whereby compounds in the droplet compete for a limited number of charged sites at the surface. As such, it is capacity-limited, and LC–MS-based techniques must be carefully evaluated to ensure that matrix effects or coeluting drugs do not impact quantitative assay performance. In this report, we describe efforts to ameliorate such effects in the absence of an isotopically labeled internal standard. Matrix effects are highly variable and heavily dependent on the physico-chemical properties of the substance. Although there is no universal solution to their resolution, conditions at the electrospray interface can mitigate these issues. Using this approach, the LC–MS/MS assay was fully validated and limits of detection and quantitation of 0.1 and 0.5 ng/mL suvorexant were achieved in blood.

Sign in / Sign up

Export Citation Format

Share Document