Dynamic Range Of Quantification Research Articles

An ultrasound multiparametric method to quantify liver fat using magnetic resonance as standard reference.

There is an unmet need for a reliable and reproducible non-invasive measure of fatty liver content (FLC) for monitoring steatotic liver disease in clinical practice. Sonographic FLC assessment is qualitative and operator-dependent, and the dynamic quantification range of algorithms based on a single ultrasound (US) parameter is unsatisfactory. This study aims to develop and validate a new multiparametric algorithm based on B-mode images to quantify FLC using Magnetic Resonance (MR) values as standard reference. Patients with elevated liver enzymes and/or bright liver at US (N = 195) underwent FLC evaluation by MR and by US. Five US-derived quantitative features [attenuation rate(AR), hepatic renal-ratio(HR), diaphragm visualization(DV), hepatic-portal-vein-ratio(HPV), portal-vein-wall(PVW)] were combined by mixed linear/exponential regression in a multiparametric model (Steatoscore2.0). One hundred and thirty-four subjects were used for training and 61 for independent validations; score-computation underwent an inter-operator reproducibility analysis. The model is based on a mixed linear/exponential combination of 3 US parameters (AR, HR, DV), modelled by 2 equations according to AR values. The computation of FLC by Steatoscore2.0 (mean ± std, 7.91% ± 8.69) and MR (mean ± std, 8.10% ± 10.31) is highly correlated with a low root mean square error in both training/validation cohorts, respectively (R = 0.92/0.86 and RMSE = 5.15/4.62, p < .001). Steatoscore2.0 identified patients with MR-FLC≥5%/≥10% with sensitivity = 93.2%/89.4%, specificity = 86.1%/95.8%, AUROC = 0.958/0.975, respectively and correlated with MR (R = 0.92) significantly (p < .001) better than CAP (R = 0.73). Multiparametric Steatoscore2.0 measures FLC providing values highly comparable with MR. It is reliable, inexpensive, easy to use with any US equipment and qualifies to be tested in larger, prospective studies as new tool for the non-invasive screening and monitoring of FLC.

A new colorimetric paper-based detection of furfural vapor as a fuel marker

Conventional fuel dye markers used in prevention of fuel smuggling and unauthorized mixing generally require laborious sample preparation including extraction and mixing of color developer in liquid phase which also generate high waste volume. In this work, furfural is used as an economical fuel marker which can be directly detected in vapor phase without extraction providing low waste generation. The ambient vapor pressure of furfural allows adequate amount of the marker to be captured by filter paper coated with amine color developers and detected by smartphone camera for colorimetric quantification using ImageJ processing software. The color is developed by either the Stenhouse reaction or imine condensation of amine with furfural in the presence of an appropriate acid. The dynamic quantification range of furfural marker in gasoline fuel is 5–35 ppm with the detection limit at 3.10 ppm.

Development of a duplex q-PCR for the simultaneous detection of Parachlamydia acanthamoebae and Simkania negevensis in environmental and clinical samples

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction

IntroductionUnvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. ObjectiveTo develop and test a control RNA for YFV detection through real-time RT-PCR. MethodsA 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. ResultsA dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). ConclusionThe plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics.

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson's r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments.

Multicenter clinical evaluation of alinity m HBV assay performance.

Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New generations of real-time HBV DNA assay platforms provide results in less than 2-3 h, with continuous loading of specimens and true random-access capability. We examined the clinical performance of the new Alinity m HBV assay, run on the fully automated, continuous, random-access Alinity m platform, to accurately detect and quantify HBV DNA in a large series of patient samples infected with different HBV genotypes frequently encountered in clinical practice. This international, multisite study assessed the precision and reproducibility of the Alinity m HBV assay and compared its performance to four HBV assays currently in clinical use. The Alinity m HBV assay demonstrated linear quantitation of HBV DNA in plasma samples, with high precision (coefficient of variation 4.1 %-8.8 %) and reproducibility. The Alinity m HBV assay showed excellent correlation (correlation coefficients ≥0.947) with comparator HBV assays, with an overall observed bias ranging from -0.07 to 0.17 Log10 IU/mL. 97 % of quantifiable patient results were <1 Log10 IU/mL different than the respective comparator assays, with comparable results across HBV genotypes. The newly developed real-time PCR-based Alinity m HBV assay is sensitive, reproducible, and accurately quantifies HBV DNA levels from HBsAg-positive patients across the full dynamic range of quantification.

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA.

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Establishment of Simultaneous Detection and Quantitation of HCVRNA by Third Generation Intercalating Dye Real-time PCR

Introduction: Rapid quantification of hepatitis C virus is helpful in determining and monitoring of the disease progression and nature of the virus replication. The aim of the present study was to establish a fast, specific and sensitive tool for HCVRNA quantification. Materials and Methods: A total of 50 serum samples, comprising of 40 HCV-positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to real-time PCR amplification. Real-time PCR using EvaGreen dye and primers targeting a 5’UTR was carried out. Reference samples with known viral load were treated similarly to the unknown samples and used to create the standard curves. Results: Our method showed a high level of analytical specificity and accuracy, with a low limit of detection (~2 IU/ml). It yielded repeatable results with less than 4% of intra- assay variation. The assay covered a broad dynamic range of quantification, ranging from 0.34 to 6 log IU/ml. The diagnostic sensitivity, specificity, and accuracy were all 100%, indicating neither false positive nor false negative results were obtained. Conclusion: The developed real time PCR using EvaGreen dye has demonstrated a highly analytical and diagnostic performance for HCV quantification, suggesting its potential in clinical diagnosis and management.

Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux® 3μm AMP column (150 x 3.0mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50pg/mL, respectively. The linear dynamic range of quantification was 50-10000pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.

High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS) was developed for the simultaneous determination of 28 amphetamine-type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid-liquid extraction (LLE) at pH9.5 using methyl tert-butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization. All stimulants were eluted within 7minutes and baseline separation was achieved for isomeric and isobaric compounds using HILIC chromatography. Extraction efficiency was greater than 80% and matrix effect was acceptable for most stimulants. The limit of detection (LOD) was in the range of 10-50pg/mL and the lower limit of quantification (LLOQ) was in the range of 50-100pg/mL. Quadratic regression was employed for quantification and the dynamic range of quantification was 50-10000pg/mL. Confirmatory analysis criteria were established using product ion ratios and retention time. The limit of confirmation (LOC) was in the range of 20-100pg/mL. Stability study results indicated that some stimulants were unstable in equine plasma at room temperature and 4°C. However, all the stimulants studied were stable at -20°C and-80°C for the 6month study period.

Silver nanoclusters based FRET aptasensor for sensitive and selective fluorescent detection of T-2 toxin

T-2 is a trichthecenes mycotoxin responsible for severe food poisoning and toxic influences in humans and animals. Therefore, a rapid and sensitive detection of T-2 is an urgent need to protect the public health mainly caused by the consumption of contaminated agricultural products. Herein, we have prepared one pot synthesis of aptamer functionalized silver nanoclusters (apt-AgNCs) for fluorescent detection of T-2 that relies on novel fluorescence resonance energy transfer (FRET). Apt-AgNCs, act as energy donor, were applied as fluorescent sensing probe whose fluorescence was quenched by MoS2 employed as fluorescent acceptor. Introduction of target in proposed bioassay leads to desorption of Apt-AgNCs from MoS2, and recovery of fluorescence in a toxin concentration dependent-manner. A dynamic quantification range was 0.005–500 ng mL−1 while the LOD was 0.93 pg mL−1. Spiking analysis reveals the efficiency and robustness of the developed assembly, with recovery percentages ranged from 89.46% to 102.08% and 90.41% to 107.75% in maize and wheat samples, respectively. The results were cross-verified using standard commercial T-2 EISA kit. In conclusion, developed bioassay may find good utility in testing for risk assessment of T-2 toxin.

Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip.

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry.

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

Easy, Fast, and Reproducible Quantification of Cholesterol and Other Lipids in Human Plasma by Combined High Resolution MSX and FTMS Analysis

Reliable, cost-effective, and gold-standard absolute quantification of non-esterified cholesterol in human plasma is of paramount importance in clinical lipidomics and for the monitoring of metabolic health. Here, we compared the performance of three mass spectrometric approaches available for direct detection and quantification of cholesterol in extracts of human plasma. These approaches are high resolution full scan Fourier transform mass spectrometry (FTMS) analysis, parallel reaction monitoring (PRM), and novel multiplexed MS/MS (MSX) technology, where fragments from selected precursor ions are detected simultaneously. Evaluating the performance of these approaches in terms of dynamic quantification range, linearity, and analytical precision showed that the MSX-based approach is superior to that of the FTMS and PRM-based approaches. To further show the efficacy of this approach, we devised a simple routine for extensive plasma lipidome characterization using only 8 μL of plasma, using a new commercially available ready-to-spike-in mixture with 14 synthetic lipid standards, and executing a single 6 min sample injection with combined MSX analysis for cholesterol quantification and FTMS analysis for quantification of sterol esters, glycerolipids, glycerophospholipids, and sphingolipids. Using this simple routine afforded reproducible and absolute quantification of 200 lipid species encompassing 13 lipid classes in human plasma samples. Notably, the analysis time of this procedure can be shortened for high throughput-oriented clinical lipidomics studies or extended with more advanced MSALL technology (Almeida R. et al., J. Am. Soc. Mass Spectrom. 26, 133-148 [1]) to support in-depth structural elucidation of lipid molecules. Graphical Abstract ᅟ.

Bioanalytical PK support for Immunotherapeutics: The need for sensitivity combined with broad assay range - Case studies

Event Abstract Back to Event Bioanalytical PK support for Immunotherapeutics: The need for sensitivity combined with broad assay range - Case studies Christian Pieper1, Beena Punnamoottil1, Nina Juergens2, Andreas Jonas2, Michael Adler3 and Mark Spengler1* 1 Chimera Biotec, Project Management, Germany 2 Chimera Biotec, Bioanalysis, Germany 3 Chimera Biotec, Assay Development, Germany Purpose: Immunotherapeutic concepts play an increasingly important role in modern drug development. Dosing of high efficient antibodies, the most common class of immune-modulatory drugs, is challenging. Therapeutic dosing regime may vary significantly, depending on safety, binding targets, potency, clearance rate, physiological effects, and other considerations. Hence, for optimal trial support, assay range of the supporting method is key, addressing both, high and low dosing. Methods: Here we evaluate DNA-enhanced (Immuno-PCR/Imperacer) state-of-the-art bioanalytical approaches for GxP regulated study support with broad dynamic quantification range. Target-specific antibody-DNA conjugates were applied for subsequent exponential signal amplification with PCR (Polymerase Chain Reaction). Results: In analysis of case studies from the development of immunotherapeutic compounds, detection range from sub-ng/ ml up to μg/ml target concentrations in biological matrix is demonstrated. Accuracy and precision of the assays were well in accordance with current guideline requirements in assay validation. Any source sample dilution furthermore enhances assay robustness and enables compatibility with limited sample volume availability, as typical in e.g. ophthalmology. Conclusions: Imperacer technology provides sufficient sensitivity, continuous dynamic range, and GxP compliance for analysis of immunotherapeutic drugs. This analysis strategy also opens up additional applications such as polyplex quantification of multiple analytes (e.g. drug and target) from one sample and/or microsampling sample volume reduction (<10 μl for duplicate analysis). Keywords: pharmacokinetic, pk, Clinical support, immuno-PCR, IPCR, ultra sensitive immunoassays Conference: EUFEMED 2017, London, United Kingdom, 17 May - 19 May, 2017. Presentation Type: Poster Topic: EUFEMED 2017 CONFERENCE Citation: Pieper C, Punnamoottil B, Juergens N, Jonas A, Adler M and Spengler M (2019). Bioanalytical PK support for Immunotherapeutics: The need for sensitivity combined with broad assay range - Case studies. Front. Pharmacol. Conference Abstract: EUFEMED 2017. doi: 10.3389/conf.fphar.2017.62.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Aug 2017; Published Online: 25 Jan 2019. * Correspondence: Dr. Mark Spengler, Chimera Biotec, Project Management, Bremen, Germany, spengler@chimera-biotec.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Christian Pieper Beena Punnamoottil Nina Juergens Andreas Jonas Michael Adler Mark Spengler Google Christian Pieper Beena Punnamoottil Nina Juergens Andreas Jonas Michael Adler Mark Spengler Google Scholar Christian Pieper Beena Punnamoottil Nina Juergens Andreas Jonas Michael Adler Mark Spengler PubMed Christian Pieper Beena Punnamoottil Nina Juergens Andreas Jonas Michael Adler Mark Spengler Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor

Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo.

Disposable and portable electrochemical aptasensor for label free detection of aflatoxin B1 in alcoholic beverages

Aflatoxin B1 (AFB1) is the most prevalent and toxic contaminant for human beings and animals among all the aflatoxins (AFs). Additionally, AFB1 is documented as a highly carcinogenic and mutagenic contaminant in the literature. Therefore, it is of vital importance to monitor AFB1 contamination to reduce its health associated risks. In the present work, we have designed a label-free electrochemical impedimetric aptasensor for aflatoxin B1 detection. Herein, we compared the analytical performances of two aptamer sequences (seqA and seqB). The detection is based on specific recognition by the aptamer covalently-bound as compact monolayer on screen printed carbon electrodes (SPCEs) via diazonium coupling reaction. The quantification of AFB1 was achieved by using electrochemical impedance spectroscopy. A dynamic quantification range from 0.125ngmL−1 to 16ngmL−1 was obtained with both types of aptamer sequences while the detection limits were 0.12ngmL−1 and 0.25ngmL−1 for seqA and seqB respectively. For real sample applications, the developed aptasensors were demonstrated in beer and wine samples, and good recovery levels in the range of 92–102% were recorded for AFB1 detection.

Simultaneous Detection of Selected Enteric Viruses in Water Samples by Multiplex Quantitative PCR

Human rotavirus (HRV), adenovirus (HAdV), and norovirus (HNV) are the most common causes of viral gastroenteritis in humans worldwide. Application of quantitative PCR (qPCR) coupled with reverse transcription (RT), known as RT-qPCR, has greatly improved detection sensitivity for enteric viruses, but it has been mostly used in monoplex mode where only a single virus type can be quantified per assay. Here, we report the development of a simple and specific multiplex RT-qPCR assay for simultaneous quantification of rotavirus, adenovirus, and norovirus in ground water, river water, and wastewater samples. For all three virus groups, the monoplex and multiplex RT-qPCR yielded virtually identical PCR efficiency, dynamic quantification range, and detection sensitivity, indicating the reliability of the multiplex RT-qPCR assay for simultaneous quantification of three viruses. The multiplex assay also accurately quantified the target genes of a small number (40 copies per PCR) in the presence of competing viral genes of up to 104-fold more abundant. Specificity of the assay was estimated to be 100 % for all three viruses when tested against 19 common waterborne microorganisms. The results showed that our multiplex RT-qPCR assay holds considerable promise in quantifying the three enteric viruses in environmental water samples.