scholarly journals Ultrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Antonio Minopoli ◽  
Bartolomeo Della Ventura ◽  
Bohdan Lenyk ◽  
Francesco Gentile ◽  
Julian A. Tanner ◽  
...  
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Sample Pretreatment ◽  
High Specificity ◽  
Ease Of Use ◽  
Enhanced Fluorescence ◽  
High Throughput Analysis ◽  
Malaria Rapid Diagnostic Tests ◽  
Copolymer Micelle ◽  
Very High

AbstractDevelopment of plasmonic biosensors combining reliability and ease of use is still a challenge. Gold nanoparticle arrays made by block copolymer micelle nanolithography (BCMN) stand out for their scalability, cost-effectiveness and tunable plasmonic properties, making them ideal substrates for fluorescence enhancement. Here, we describe a plasmon-enhanced fluorescence immunosensor for the specific and ultrasensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH)—a malaria marker—in whole blood. Analyte recognition is realized by oriented antibodies immobilized in a close-packed configuration via the photochemical immobilization technique (PIT), with a top bioreceptor of nucleic acid aptamers recognizing a different surface of PfLDH in a sandwich conformation. The combination of BCMN and PIT enabled maximum control over the nanoparticle size and lattice constant as well as the distance of the fluorophore from the sensing surface. The device achieved a limit of detection smaller than 1 pg/mL (<30 fM) with very high specificity without any sample pretreatment. This limit of detection is several orders of magnitude lower than that found in malaria rapid diagnostic tests or even commercial ELISA kits. Thanks to its overall dimensions, ease of use and high-throughput analysis, the device can be used as a substrate in automated multi-well plate readers and improve the efficiency of conventional fluorescence immunoassays.

scholarly journals Randomly positioned gold nanoparticles as fluorescence enhancers in apta-immunosensor for malaria test

2021 ◽  
Vol 188 (3) ◽  
Author(s):  
Antonio Minopoli ◽  
Bartolomeo Della Ventura ◽  
Raffaele Campanile ◽  
Julian A. Tanner ◽  
Andreas Offenhäusser ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Self Assembly ◽  
Whole Blood ◽  
Sample Pretreatment ◽  
High Specificity ◽  
Enhanced Fluorescence ◽  
High Throughput Analysis ◽  
New Approach ◽  
Fluorescence Amplification

AbstractA plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays. Graphical abstract

scholarly journals A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

2017 ◽  
Author(s):  
Yanan Du ◽  
Xiao Zhao ◽  
Binan Zhao ◽  
Yan Xu ◽  
Wei Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
Pathogenic Bacteria ◽  
Limit Of Detection ◽  
High Specificity ◽  
High Throughput Analysis ◽  
Fluorescent Intensity ◽  
Fluorescence Spectrophotometry ◽  
Gm Maize ◽  
Specificity And Sensitivity ◽  
High Throughput Detection

AbstractHere we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high -throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluorensence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co -amplified products with different fluorophores could be simultaneously analyzed to achieve high -throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w / w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.

scholarly journals PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S57-S57
Author(s):  
Edgar Ong ◽  
Ruo Huang ◽  
Richard Kirkland ◽  
Michael Hale ◽  
Larry Mimms
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Quantitative Detection ◽  
Therapeutic Drug ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Assay Precision

Abstract Introduction A fast (&lt;5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “&gt;ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of &lt;2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

scholarly journals Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anita G. Amin ◽  
Prithwiraj De ◽  
Barbara Graham ◽  
Roger I. Calderon ◽  
Molly F. Franke ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sample Pretreatment ◽  
Positive Association ◽  
Proteinase K ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Smear Negative ◽  
Tuberculostearic Acid ◽  
Negative Sputum ◽  
Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

Evaluation of the first immunosuppressive drug assay available on a fully automated LC-MS/MS-based clinical analyzer suggests a new era in laboratory medicine

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Sebastian Hörber ◽  
Andreas Peter ◽  
Rainer Lehmann ◽  
Miriam Hoene
Keyword(s):  
High Specificity ◽  
Turnaround Time ◽  
Immunosuppressive Drugs ◽  
Immunosuppressive Drug ◽  
Ease Of Use ◽  
Analytical Performance ◽  
University Hospital ◽  
Therapeutic Monitoring ◽  
Mean Deviation ◽  
Liquid Chromatography Tandem Mass

AbstractObjectivesDue to its high specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard in diagnostic areas such as therapeutic monitoring of immunosuppressive drugs (ISDs). However, many laboratories still rely on immunoassays for ISD quantification in a tradeoff between analytical performance and the advantages of fully automated analyzers – shorter turnaround times, greater ease of use, and 24/7 availability.MethodsThe LC-MS/MS-based Thermo Scientific™ Cascadion™ SM Immunosuppressant Panel was evaluated for >6 months in the routine laboratory of a university hospital. We assessed the analytical performance of the panel and compared it to conventional LC-MS/MS as well as to immunoassays (cyclosporine A, sirolimus, tacrolimus (Siemens) and everolimus (Thermo Fisher)). In addition, both ISD panel and Cascadion analyzer were scrutinized with regards to, e.g., turnaround time, usability, and robustness.ResultsAll ISDs showed high linearity and precision (CV≤6%) and a good correlation with conventional LC-MS/MS. The mean deviation to the immunoassays was 17–19% and negative for all ISDs except everolimus with a positive 19% bias. No weak points were revealed when challenging assay and system with, e.g., high haematocrit, sedimented whole blood or priority samples. The Cascadion integrated well into our 24/7 routine and could easily be operated simultaneously with several other analyzers by technical staff without LC-MS experience.ConclusionsThe ISD panel showed excellent analytical performance and demonstrated that a fully automated LC-MS-based analysis starting from primary samples is feasible, suggesting that LC-MS could become an integral part of 24/7 diagnostics in the near future.

A benzothiazole-based fluorescence turn-on sensor for copper(II)

2021 ◽  
Author(s):  
Gyeongjin Kim ◽  
Donghwan Choi ◽  
Cheal Kim
Keyword(s):  
Charge Transfer ◽  
Limit Of Detection ◽  
Theoretical Calculations ◽  
Enhanced Fluorescence ◽  
Turn On ◽  
Fluorescent Response ◽  
Chelation Enhanced Fluorescence ◽  
Fluorescence Turn On ◽  
Internal Charge Transfer ◽  
Internal Charge

Abstract A new benzothiazole-based chemosensor BTN (1-((Z)-(((E)-3-methylbenzo[d]thiazol-2(3H)-ylidene)hydrazono)methyl)naphthalen-2-ol) was synthesized for the detection of Cu2+. BTN could detect Cu2+ with “off-on” fluorescent response from colorless to yellow irrespective of presence of other cations. Limit of detection for Cu2+ was determined to be 3.3 µM. Binding ratio of BTN and Cu2+ turned out to be a 1:1 with the analysis of Job plot and ESI-MS. Sensing feature of Cu2+ by BTN was explained with theoretical calculations, which might be owing to internal charge transfer and chelation-enhanced fluorescence processes.

Cds nanocrystal enhanced TiO2photoelectrochemical aptasensor for sensitive detection of cytochrome c

2021 ◽  
Vol 11 (11) ◽  
pp. 1774-1780
Author(s):  
Shanji Fan ◽  
Hong Huang ◽  
Hong Chen ◽  
Jiachi Xu ◽  
Zecheng Hu ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Limit Of Detection ◽  
Specific Binding ◽  
High Sensitivity ◽  
High Specificity ◽  
Linear Range ◽  
Current Intensity ◽  
Sensitive Detection ◽  
Layer Adsorption ◽  
Ionic Layer

A CdS nanocrystal enhanced TiO2 nanotubes (CdS@TiO2 NATs) photoelectrode was prepared via successive ionic layer adsorption and reaction (SILAR) of CdS on the surface of TiO2 NATs. A HS-aptamer owing a specific binding toward cytochrome c was modified onto the CdS@TiO2 NATs, which resulting a decrease in the photoelectrical current intensity. Cytochrome c is therefore quantified based on the decrease in photoelectrical current. High specificity and high sensitivity were obtained with a linear range from 3 pM to 80 nM, and a limit of detection of 2.53 pM.

Two Orders of Magnitude Improvement in the Detection Limit of Droplet-Based Micro-Magnetofluidics with Planar Hall Effect Sensors

2021 ◽  
Vol 6 (1) ◽  
pp. 47
Author(s):  
Julian Schütt ◽  
Rico Illing ◽  
Oleksii Volkov ◽  
Tobias Kosub ◽  
Pablo Nicolás Granell ◽  
...  
Keyword(s):  
Detection Limit ◽  
Hall Effect ◽  
State Of The Art ◽  
Limit Of Detection ◽  
Research Field ◽  
High Throughput Analysis ◽  
Planar Hall Effect ◽  
Biologically Relevant ◽  
Sensing Platform ◽  
Order Of Magnitude

The detection, manipulation, and tracking of magnetic nanoparticles is of major importance in the fields of biology, biotechnology, and biomedical applications as labels as well as in drug delivery, (bio-)detection, and tissue engineering. In this regard, the trend goes towards improvements of existing state-of-the-art methodologies in the spirit of timesaving, high-throughput analysis at ultra-low volumes. Here, microfluidics offers vast advantages to address these requirements, as it deals with the control and manipulation of liquids in confined microchannels. This conjunction of microfluidics and magnetism, namely micro-magnetofluidics, is a dynamic research field, which requires novel sensor solutions to boost the detection limit of tiny quantities of magnetized objects. We present a sensing strategy relying on planar Hall effect (PHE) sensors in droplet-based micro-magnetofluidics for the detection of a multiphase liquid flow, i.e., superparamagnetic aqueous droplets in an oil carrier phase. The high resolution of the sensor allows the detection of nanoliter-sized superparamagnetic droplets with a concentration of 0.58 mg cm−3, even when they are only biased in a geomagnetic field. The limit of detection can be boosted another order of magnitude, reaching 0.04 mg cm−³ (1.4 million particles in a single 100 nL droplet) when a magnetic field of 5 mT is applied to bias the droplets. With this performance, our sensing platform outperforms the state-of-the-art solutions in droplet-based micro-magnetofluidics by a factor of 100. This allows us to detect ferrofluid droplets in clinically and biologically relevant concentrations, and even in lower concentrations, without the need of externally applied magnetic fields.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

scholarly journals Occurrence of adenovirus infection and clinical impact in paediatric stem cell transplant recipients

2016 ◽  
Vol 31 (3) ◽  
Author(s):  
Gabriele Bianco ◽  
Cristina Costa ◽  
Andrea Piceghello ◽  
Francesca Sidoti ◽  
Mareva Giacchino ◽  
...  
Keyword(s):  
Stem Cell ◽  
Whole Blood ◽  
Limit Of Detection ◽  
Antiviral Agent ◽  
Clinical Impact ◽  
Cell Transplant ◽  
Adenovirus Infection ◽  
Hematopoietic Stem ◽  
Organ Specific ◽  
Blood Specimens

In this study, the occurrence and clinical impact of adenovirus (AdV) infection was investigated in paediatric hematopoietic stem cell transplantation (HSCT) recipients. A number of 603 specimens (including whole blood, respiratory and other samples) from 181 patients were tested by real-time polymerase chain reaction; clinical outcome was investigated. Overall, 118/603 (19.6%) specimens from 21/181 (11.6%) patients resulted positive to AdV (including 17.3, 29.9, 17.6, and 15.8% of total number of whole blood, respiratory, urine and other specimens, respectively). On whole blood specimens, viral loads ranged from &lt;600 (limit of detection) to &gt;5×10<sup>6</sup> copies/mL, with a median value 2×104. Multiple specimens were positive in patients in which viral load on whole blood was high. Adenoviral positivity on whole blood was associated to poor prognosis, as death occurred in three of ten (30%) patients with persistent positivity on whole blood specimens, also despite the administration of an antiviral agent (cidofovir). Adenovirus infection can account for systemic and/or organ-specific signs/symptoms in approximately 10% of paediatric HSCT recipients. At moment, there is no indication for routine monitor of AdV in these patients, although AdV aetiology of infectious transplant complications should be taken in account.

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