scholarly journals A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

2022 ◽  
Vol 12 ◽  
Author(s):  
Katharina Radakovics ◽  
Claire Battin ◽  
Judith Leitner ◽  
Sabine Geiselhart ◽  
Wolfgang Paster ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Ectopic Expression ◽  
High Sensitivity ◽  
High Specificity ◽  
Reporter System ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Definition Of ◽  
The Individual ◽  
Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

scholarly journals Laboratory and Clinical Evaluation of DNA Microarray for the Detection of Carbapenemase Genes in Gram-Negative Bacteria from Hospitalized Patients

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Yi Song ◽  
Fengna Dou ◽  
Sha He ◽  
Yu Zhou ◽  
Qiqi Liu
Keyword(s):  
Dna Microarray ◽  
High Throughput ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Specificity And Sensitivity ◽  
Global Threat

Background. The prevalence of a variety of carbapenemases in Gram-negative bacteria (GNB) has posed a global threat on clinical control and management. Monitoring and controlling the carbapenemase-producing GNB became imperative tasks for many healthcare centers. The aim of this study was to develop a high-throughput, specific, sensitive, and rapid DNA microarray-based method for the diagnosis, phenotypic confirmation, and molecular epidemiological study of carbapenemase genes. Methods. We targeted a panel of eight carbapenemase genes, including blaKPC, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-51, blaIMP, blaVIM, and blaDIM for detection. Ultrasensitive chemiluminescence (CL) detection method was developed and used to simultaneously detect eight carbapenemase genes, and plasmids were established as positive or limit of detection (LOD) reference materials. Antibiotic susceptibility was determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines in order to screen clinical isolates resistant to carbapenem antibiotics as well as Sanger sequencing which was used to confirm the reliability of the results presented by DNA microarray. Results. Eight carbapenemase genes could be detected with high sensitivity and specificity. The absolute LOD of this strategy to detect serially diluted plasmids of eight carbapenemase genes was 102- 103copies/μL. Then, 416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests. Compared with Sanger sequencing, a specificity and sensitivity of 100% were recorded for blaNDM-1, blaIMP, blaVIM, and blaDIM genes. The specificity for blaKPC, blaOXA-23, blaOXA-48, and blaOXA-51 genes was 100% and the sensitivity was 98.5%, 97.6%, 95.7%, and 97.9%, respectively. The overall consistency rate between the sequencing and microarray is 97.8%. Conclusions. The proposed ultrasensitive CL imaging DNA hybridization has high specificity, sensitivity, and reproducibility and could detect and differentiate clinical specimens that carried various carbapenemase genes, suggesting that the method can conveniently be customized for high-throughput detection of the carbapenemase-producing GNB and can be easily adapted for various clinical applications.

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.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

A Preliminary Evaluation of a Prototype Western Blot Confirmatory Test Kit for Syphilis

1994 ◽  
Vol 5 (6) ◽  
pp. 409-414 ◽  
Author(s):  
H Young ◽  
P J Walker ◽  
D Merry ◽  
A Mifsud
Keyword(s):  
Western Blot ◽  
False Positive ◽  
High Sensitivity ◽  
High Specificity ◽  
Confirmatory Test ◽  
Preliminary Evaluation ◽  
Serological Tests ◽  
Western Blot Test ◽  
Specificity And Sensitivity ◽  
Test Kit

A prototype Western blot kit was evaluated as a confirmatory test for syphilis using 131 sera characterized by other serological tests for syphilis. There were 114 treponemal sera (including 94 cases of early syphilis, 83 of which were untreated) and 17 non-treponemal problem sera (11 gave false positive reactions on screening with the TmpA recombinant antigen enzyme immunoassay (EIA), 3 gave false positive fluorescent treponemal antibody absorbed (FTA-abs) tests, and 3 false positive Captia Syphilis G EIA results). Based on the manufacturer's criteria of reactivity in multiple bands for designating a positive result the Western blot test gave a sensitivity of 99.1% (113/114) and a specificity of 88.2% (15/17) when indeterminate reactions were scored positive and 98.2% (112/114) and 100% (17/17) when indeterminate reactions were scored negative. Sensitivity was high in both treated and untreated infection. Corresponding sensitivities for the TPHA and FTA-abs when equivocal reactions were scored negative were 97.5% (111/114) and 99.1% (113/114). The high sensitivity of the FTA-abs in this study is probably due to the large number of untreated primary infections. Our results with the Western blot, confirm earlier studies using ‘in-house’ test systems and, support a role for a commercial Western blot test in the confirmatory diagnosis of syphilis. Further studies are required to confirm the high specificity and sensitivity of the kit in a larger series including a wider variety of non-treponemal cases as well as patients with untreated and treated infection.

scholarly journals Rapid-Response and Highly Sensitive Boronate Derivative-Based Fluorescence Probe for Detecting H2O2 in Living Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Muthusamy Selvaraj ◽  
Kanagaraj Rajalakshmi ◽  
Yun-Sik Nam ◽  
Yeonhee Lee ◽  
Byoung Chan Kim ◽  
...  
Keyword(s):  
Functional Group ◽  
Fluorescence Probe ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Rapid Identification ◽  
Oxygen Species ◽  
Human Breast ◽  
Highly Sensitive ◽  
Whatman Paper ◽  
Mcf 7

Intracellular H2O2 monitoring is important and has driven researchers to pursue advancements for the rapid identification of H2O2, since H2O2 is short-lived in cell lines. An arylboronate derivative has been investigated as a chemospecific fluorescence recognition agent for H2O2. Triphenylimidazoleoxadiazolephenyl (TPIOP) boronate was contrived as a novel candidate for the rapid and sensitive recognition of H2O2. The probe was conjugated using the TPIOP functional group acting as an excellent fluorescent enhancer. The TPIOP group stimulated the polarization of C–B bond due to its extended π-conjugation, which included heteroatoms, and induced the production of rapid signal because of the highly polar C–B bond along with the corresponding boronate unit. While H2O2 reacts with TPIOP boronate, its nucleophilic addition to the boron generates a charged tetrahedral boronate complex, and then the C–B bond migrates toward one of the electrophilic peroxide oxygen atoms. The resulting boronate ester is then hydrolyzed by water into a phenol, which significantly enhances fluorescence through aggregation-induced emission. The TPIOP boronate probe responded to H2O2 rapidly, within 2 min, and exhibited high sensitivity with a limit of detection of 8 nM and a 1000-fold selectivity in the presence of other reactive oxygen species. Therefore, the developed TPIOP boronate chemodosimeter was successfully utilized to visualize and quantify intracellular H2O2 from human breast cancer (MCF-7) cells, as well as gaseous and aqueous H2O2 from environmental samples using Whatman paper strips coated with TPIOP boronate.

scholarly journals Flexible and Highly Sensitive Pressure Sensors Based on Microstructured Carbon Nanowalls Electrodes

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 496 ◽  
Author(s):  
Xi Zhou ◽  
Yongna Zhang ◽  
Jun Yang ◽  
Jialu Li ◽  
Shi Luo ◽  
...  
Keyword(s):  
Pressure Sensor ◽  
High Performance ◽  
Limit Of Detection ◽  
Pressure Sensors ◽  
High Sensitivity ◽  
Physiological Signals ◽  
Healthcare Applications ◽  
Highly Sensitive ◽  
Carbon Nanowalls ◽  
Pressure Regime

Wearable pressure sensors have attracted widespread attention in recent years because of their great potential in human healthcare applications such as physiological signals monitoring. A desirable pressure sensor should possess the advantages of high sensitivity, a simple manufacturing process, and good stability. Here, we present a highly sensitive, simply fabricated wearable resistive pressure sensor based on three-dimensional microstructured carbon nanowalls (CNWs) embedded in a polydimethylsiloxane (PDMS) substrate. The method of using unpolished silicon wafers as templates provides an easy approach to fabricate the irregular microstructure of CNWs/PDMS electrodes, which plays a significant role in increasing the sensitivity and stability of resistive pressure sensors. The sensitivity of the CNWs/PDMS pressure sensor with irregular microstructures is as high as 6.64 kPa−1 in the low-pressure regime, and remains fairly high (0.15 kPa−1) in the high-pressure regime (~10 kPa). Both the relatively short response time of ~30 ms and good reproducibility over 1000 cycles of pressure loading and unloading tests illustrate the high performance of the proposed device. Our pressure sensor exhibits a superior minimal limit of detection of 0.6 Pa, which shows promising potential in detecting human physiological signals such as heart rate. Moreover, it can be turned into an 8 × 8 pixels array to map spatial pressure distribution and realize array sensing imaging.

scholarly journals DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

2015 ◽  
Vol 8 ◽  
pp. MBI.S29736 ◽  
Author(s):  
Kenjiro Nagamine ◽  
Guo-Chiuan Hung ◽  
Bingjie Li ◽  
Shyh-Ching Lo
Keyword(s):  
Safety Concern ◽  
Rapid Development ◽  
High Sensitivity ◽  
High Specificity ◽  
Clostridium Tetani ◽  
Specificity And Sensitivity ◽  
Wide Range ◽  
Pcr Assays ◽  
Primer Sets ◽  
Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

scholarly journals Novel Highly Sensitive Protein Sensors Based on Tapered Optical Fibres Modified with Au-Based Nanocoatings

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Aitor Urrutia ◽  
Kartheka Bojan ◽  
Leonel Marques ◽  
Kevin Mullaney ◽  
Javier Goicoechea ◽  
...  
Keyword(s):  
Binding Constant ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Optical Fibres ◽  
Layer By Layer ◽  
Measured Concentration ◽  
Au Nps ◽  
Layer Deposition ◽  
Highly Sensitive ◽  
Protein Sensors

Novel protein sensors based on tapered optical fibres modified with Au coatings deposited using two different procedures are proposed. Au-based coatings are deposited onto a nonadiabatic tapered optical fibre using (i) a novel facile method composed of layer-by-layer deposition consisting of polycation (poly(allylamine hydrochloride), PAH) and negatively charged SiO2nanoparticles (NPs) followed by the deposition of the charged Au NPs and (ii) the sputtering technique. The Au NPs and Au thin film surfaces are then modified with biotin in order to bind streptavidin (SV) molecules and detect them. The sensing principle is based on the sensitivity of the transmission spectrum of the device to changes in the refractive index of the coatings induced by the SV binding to the biotin. Both sensors showed high sensitivity to SV, with the lowest measured concentration levels below 2.5 nM. The calculated binding constant for the biotin-SV pair was2.2×10-11 M−1when a tapered fibre modified with the LbL method was used, with a limit of detection (LoD) of 271 pM. The sensor formed using sputtering had a binding constant of1.01×10-10 M−1with a LoD of 806 pM. These new structures and their simple fabrication technique could be used to develop other biosensors.

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