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Article ; Online: Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Ferreira, Lauren / Flanagan, Shane Patrick / Fogel, Ronen / Limson, Janice Leigh

2024 Volume 19, Issue 2, Page(s) e0295673

Abstract: Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which ... ...

Abstract	Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the β-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, β1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the β-subunit of hCG and another to the β1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the β-subunit and the (presumably) β1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.
MeSH term(s)	Pregnancy ; Female ; Humans ; Epitopes ; Chorionic Gonadotropin, beta Subunit, Human ; Chorionic Gonadotropin/metabolism ; Peptide Fragments ; Immunoassay ; Neoplasms ; Antibodies, Monoclonal
Chemical Substances	Epitopes ; Chorionic Gonadotropin, beta Subunit, Human ; Chorionic Gonadotropin ; Peptide Fragments ; Antibodies, Monoclonal
Language	English
Publishing date	2024-02-23
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0295673
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Overview of Diagnostic Methods, Disease Prevalence and Transmission of Mpox (Formerly Monkeypox) in Humans and Animal Reservoirs.

Chauhan, Ravendra P / Fogel, Ronen / Limson, Janice

Microorganisms

2023 Volume 11, Issue 5

Abstract: Mpox-formerly monkeypox-is a re-emerging zoonotic virus disease, with large numbers of human cases reported during multi-country outbreaks in 2022. The close similarities in clinical symptoms that Mpox shares with many orthopoxvirus (OPXV) diseases make ... ...

Abstract	Mpox-formerly monkeypox-is a re-emerging zoonotic virus disease, with large numbers of human cases reported during multi-country outbreaks in 2022. The close similarities in clinical symptoms that Mpox shares with many orthopoxvirus (OPXV) diseases make its diagnosis challenging, requiring laboratory testing for confirmation. This review focuses on the diagnostic methods used for Mpox detection in naturally infected humans and animal reservoirs, disease prevalence and transmission, clinical symptoms and signs, and currently known host ranges. Using specific search terms, up to 2 September 2022, we identified 104 relevant original research articles and case reports from NCBI-PubMed and Google Scholar databases for inclusion in the study. Our analyses observed that molecular identification techniques are overwhelmingly being used in current diagnoses, especially real-time PCR (3982/7059 cases;
Language	English
Publishing date	2023-04-30
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2720891-6
ISSN	2076-2607
ISSN	2076-2607
DOI	10.3390/microorganisms11051186
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Article: Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa.

Chauhan, Ravendra P / Fogel, Ronen / Limson, Janice

Microorganisms

2023 Volume 11, Issue 11

Abstract: White spot syndrome virus is a highly contagious pathogen affecting shrimp farming worldwide. The host range of this virus is primarily limited to crustaceans, such as shrimps, crabs, prawns, crayfish, and lobsters; however, several species of non- ... ...

Abstract	White spot syndrome virus is a highly contagious pathogen affecting shrimp farming worldwide. The host range of this virus is primarily limited to crustaceans, such as shrimps, crabs, prawns, crayfish, and lobsters; however, several species of non-crustaceans, including aquatic insects, piscivorous birds, and molluscs may serve as the vectors for ecological dissemination. The present study was aimed at studying the faecal virome of domestic chickens (
Language	English
Publishing date	2023-11-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720891-6
ISSN	2076-2607
ISSN	2076-2607
DOI	10.3390/microorganisms11112802
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Article ; Online: A microfluidic paper analytical device using capture aptamers for the detection of PfLDH in blood matrices.

Ogunmolasuyi, Adewoyin Martin / Fogel, Ronen / Hoppe, Heinrich / Goldring, Dean / Limson, Janice

Malaria journal

2022 Volume 21, Issue 1, Page(s) 174

Abstract: Background: The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the ... ...

Abstract	Background: The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker-Plasmodium falciparum lactate dehydrogenase (rPfLDH)-is reported in this study. Methods: Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results: Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a K Conclusion: The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.
MeSH term(s)	Aptamers, Nucleotide/metabolism ; Biomarkers/metabolism ; Humans ; L-Lactate Dehydrogenase/metabolism ; Malaria/diagnosis ; Malaria, Falciparum/diagnosis ; Microfluidics ; Plasmodium ; Plasmodium falciparum ; Streptavidin
Chemical Substances	Aptamers, Nucleotide ; Biomarkers ; Streptavidin (9013-20-1) ; L-Lactate Dehydrogenase (EC 1.1.1.27)
Language	English
Publishing date	2022-06-07
Publishing country	England
Document type	Journal Article
ZDB-ID	2091229-8
ISSN	1475-2875 ; 1475-2875
ISSN (online)	1475-2875
ISSN	1475-2875
DOI	10.1186/s12936-022-04187-6
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Article: Nonspecific nuclear uptake of anti-MUC1 aptamers by dead cells: the role of cell viability monitoring in aptamer targeting of membrane-bound protein cancer biomarkers

Flanagan, Shane Patrick / Fogel, Ronen / Edkins, Adrienne Lesley / Ho, Lance St. John / Limson, Janice

Analytical methods. 2021 Mar. 11, v. 13, no. 9

2021

Abstract: Most aptamers targeting cell-expressed antigens are intended for in vivo application, however, these sequences are commonly generated in vitro against synthetic oligopeptide epitopes or recombinant proteins. As these in vitro analogues frequently do not ... ...

Abstract	Most aptamers targeting cell-expressed antigens are intended for in vivo application, however, these sequences are commonly generated in vitro against synthetic oligopeptide epitopes or recombinant proteins. As these in vitro analogues frequently do not mimic the in vivo target within an endogenous environment, the evolved aptamers are often prone to nonspecific binding. The presence of dead cells and cellular debris further complicate aptamer targeting, due to their high nonspecific affinities to single-stranded DNA. Despite these known limitations, assessment of cell viability and/or the removal of dead cells is rarely applied as part of the methodology during in vivo testing of aptamer binding. Furthermore, the extent and route(s) by which dead cells uptake existing aptamers remains to be determined in the literature. For this purpose, the previously reported aptamer sequences 5TR1, 5TR4, 5TRG2 and S22 – enriched against the MUC1 tumour marker of the mucin glycoprotein family – were used as model sequences to evaluate the influence of cell viability and the presence of nontarget cell-expressed protein on aptamer binding to the MUC1 expressing human cancer cell lines MCF-7, Hs578T, SW480, and SW620. From fluorescence microscopy analysis, all tested aptamers demonstrated extensive nonspecific uptake within the nuclei of dead cells with compromised membrane integrities. Using fluorescent-activated cell sorting (FACS), the inclusion of excess double-stranded DNA as a blocking agent showed no effect on nonspecific aptamer uptake by dead cells. Further nonspecific binding to cell-membrane bound and intracellular protein was evident for each aptamer sequence, as assessed by southwestern blotting and FACS. These factors likely contributed to the ∼120-fold greater binding response of the 5TR1 aptamer to dead MCF-7 cells over equivalent live cell populations. The identification of dead cells and cellular debris using viability stains and the subsequent exclusion of these cells from FACS analysis was identified as an essential requirement for the evaluation of aptamer binding specificity to live cell populations of the cancer cell lines MCF-7, Hs578T and SW480. The research findings stress the importance of dead cell uptake and more comprehensive cell viability screening to validate novel aptamer sequences for diagnostic and therapeutic application.
Keywords	biomarkers ; cell viability ; epitopes ; fluorescence microscopy ; humans ; mucins ; neoplasm cells ; neoplasms ; oligonucleotides ; oligopeptides ; single-stranded DNA ; therapeutics
Language	English
Dates of publication	2021-0311
Size	p. 1191-1203.
Publishing place	The Royal Society of Chemistry
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	2515210-5
ISSN	1759-9679 ; 1759-9660
ISSN (online)	1759-9679
ISSN	1759-9660
DOI	10.1039/d0ay01878c
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Article ; Online: Generation and screening of histamine-specific aptamers for application in a novel impedimetric aptamer-based sensor.

John Ho, Lance St / Fogel, Ronen / Limson, Janice Leigh

Talanta

2019 Volume 208, Page(s) 120474

Abstract: Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work ... ...

Abstract	Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work reports on the isolation of single-stranded DNA-based, histamine-targeting aptamers generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and the characterisation of these candidates via bioinformatics analysis. Aptamer binding affinity was determined by magnetic bead-based enzyme linked oligonucleotide assays, followed by the detection of unmodified histamine at a physiological pH via electrochemical impedance spectroscopy (EIS). Aptamer H47 demonstrated the lowest apparent binding affinity (72.8 ± 13.9 nmol L
Language	English
Publishing date	2019-10-16
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1500969-5
ISSN	1873-3573 ; 0039-9140
ISSN (online)	1873-3573
ISSN	0039-9140
DOI	10.1016/j.talanta.2019.120474
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Article ; Online: Developing Biosensors in Developing Countries: South Africa as a Case Study.

Fogel, Ronen / Limson, Janice

Biosensors

2016 Volume 6, Issue 1

Abstract: A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. ... ...

Abstract	A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. Many of the reported publications centre on fundamental research into the signal transduction method, using model biorecognition elements, in line with international trends. Other research in this field is spread across several areas including: the application of nanotechnology; the identification and validation of biomarkers; development and testing of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanine. Biosensor targets commonly featured were pesticides and metals. Areas of regional import to sub-Saharan Africa, such as HIV/AIDs and tuberculosis diagnosis, are also apparent in a review of the available literature. Irrespective of the targets, the challenge to the effective deployment of such sensors remains shaped by social and economic realities such that the requirements thereof are for low-cost and universally easy to operate devices for field settings. While it is difficult to disentangle the intertwined roles of national policy, grant funding availability and, certainly, of global trends in shaping areas of emphasis in research, most notable is the strong role that nanotechnology, and to a certain extent biotechnology, plays in research regarding biosensor construction. Stronger emphasis on collaboration between scientists in theoretical modelling, nanomaterials application and or relevant stakeholders in the specific field (e.g., food or health monitoring) and researchers in biosensor design may help evolve focused research efforts towards development and deployment of low-cost biosensors.
MeSH term(s)	Biosensing Techniques/methods ; Developing Countries ; HIV Infections/diagnosis ; Humans ; Nanotechnology ; South Africa ; Tuberculosis/diagnosis
Language	English
Publishing date	2016-02-02
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2662125-3
ISSN	2079-6374 ; 2079-6374
ISSN (online)	2079-6374
ISSN	2079-6374
DOI	10.3390/bios6010005
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Article ; Online: Nonspecific nuclear uptake of anti-MUC1 aptamers by dead cells: the role of cell viability monitoring in aptamer targeting of membrane-bound protein cancer biomarkers.

Flanagan, Shane Patrick / Fogel, Ronen / Edkins, Adrienne Lesley / Ho, Lance St John / Limson, Janice

Analytical methods : advancing methods and applications

2021 Volume 13, Issue 9, Page(s) 1191–1203

Abstract	Most aptamers targeting cell-expressed antigens are intended for in vivo application, however, these sequences are commonly generated in vitro against synthetic oligopeptide epitopes or recombinant proteins. As these in vitro analogues frequently do not mimic the in vivo target within an endogenous environment, the evolved aptamers are often prone to nonspecific binding. The presence of dead cells and cellular debris further complicate aptamer targeting, due to their high nonspecific affinities to single-stranded DNA. Despite these known limitations, assessment of cell viability and/or the removal of dead cells is rarely applied as part of the methodology during in vivo testing of aptamer binding. Furthermore, the extent and route(s) by which dead cells uptake existing aptamers remains to be determined in the literature. For this purpose, the previously reported aptamer sequences 5TR1, 5TR4, 5TRG2 and S22 - enriched against the MUC1 tumour marker of the mucin glycoprotein family - were used as model sequences to evaluate the influence of cell viability and the presence of nontarget cell-expressed protein on aptamer binding to the MUC1 expressing human cancer cell lines MCF-7, Hs578T, SW480, and SW620. From fluorescence microscopy analysis, all tested aptamers demonstrated extensive nonspecific uptake within the nuclei of dead cells with compromised membrane integrities. Using fluorescent-activated cell sorting (FACS), the inclusion of excess double-stranded DNA as a blocking agent showed no effect on nonspecific aptamer uptake by dead cells. Further nonspecific binding to cell-membrane bound and intracellular protein was evident for each aptamer sequence, as assessed by southwestern blotting and FACS. These factors likely contributed to the ∼120-fold greater binding response of the 5TR1 aptamer to dead MCF-7 cells over equivalent live cell populations. The identification of dead cells and cellular debris using viability stains and the subsequent exclusion of these cells from FACS analysis was identified as an essential requirement for the evaluation of aptamer binding specificity to live cell populations of the cancer cell lines MCF-7, Hs578T and SW480. The research findings stress the importance of dead cell uptake and more comprehensive cell viability screening to validate novel aptamer sequences for diagnostic and therapeutic application.
MeSH term(s)	Biomarkers, Tumor/genetics ; Cell Survival ; DNA, Single-Stranded ; Humans ; MCF-7 Cells ; Membrane Proteins ; Neoplasms
Chemical Substances	Biomarkers, Tumor ; DNA, Single-Stranded ; Membrane Proteins
Language	English
Publishing date	2021-02-19
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2515210-5
ISSN	1759-9679 ; 1759-9660
ISSN (online)	1759-9679
ISSN	1759-9660
DOI	10.1039/d0ay01878c
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Article ; Online: Generation and screening of histamine-specific aptamers for application in a novel impedimetric aptamer-based sensor

John Ho, Lance St / Fogel, Ronen / Limson, Janice Leigh

Talanta. 2020 Feb., v. 208 p.120474-

2020

Abstract	Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work reports on the isolation of single-stranded DNA-based, histamine-targeting aptamers generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and the characterisation of these candidates via bioinformatics analysis. Aptamer binding affinity was determined by magnetic bead-based enzyme linked oligonucleotide assays, followed by the detection of unmodified histamine at a physiological pH via electrochemical impedance spectroscopy (EIS). Aptamer H47 demonstrated the lowest apparent binding affinity (72.8 ± 13.9 nmol L⁻¹) towards bead immobilised histamine. When immobilised to a gold surface, H47 demonstrated the largest biosensor response (ΔRcₜ = 6.83 ± 2.00) compared to other single-stranded DNA sequences in the presence of dissolved histamine. The H47 EIS aptasensor also displayed a highly selective, concentration-dependent response towards histamine (linear range = 1 μmol L⁻¹ - 5 mmol L⁻¹), compared to other similar small molecules. Possessing an apparent binding affinity, limit of detection and limit of quantification of 7.80 ± 1.70 mmol L⁻¹, 4.83 mmol L⁻¹ and 16.08 mmol L⁻¹, respectively, the H47 EIS aptasensor demonstrates promise towards the development of aptasensors in applications which require the rapid detection of histamine in solution.
Keywords	aptasensors ; bioinformatics ; biomarkers ; detection limit ; dielectric spectroscopy ; electrochemistry ; enzymes ; fluorescence ; food quality ; gold ; histamine ; magnetism ; oligonucleotides ; pH ; quality control ; rapid methods ; single-stranded DNA ; systematic evolution of ligands by exponential enrichment ; Aptamer ; SELEX ; Aptasensor ; EIS ; ELONA
Language	English
Dates of publication	2020-02
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	1500969-5
ISSN	1873-3573 ; 0039-9140
ISSN (online)	1873-3573
ISSN	0039-9140
DOI	10.1016/j.talanta.2019.120474
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Article ; Online: Unsubstituted metallophthalocyanine catalysts for the removal of endocrine disrupting compounds using H

Kruid, Jan / Fogel, Ronen / Limson, Janice

Environmental science and pollution research international

2018 Volume 25, Issue 32, Page(s) 32346–32357

Abstract: Advanced oxidation processes have become increasingly important to treat non-biodegradable compounds entering environmental waters. In recent decades, water-soluble metallophthalocyanines have been shown to catalyse ... ...

Abstract	Advanced oxidation processes have become increasingly important to treat non-biodegradable compounds entering environmental waters. In recent decades, water-soluble metallophthalocyanines have been shown to catalyse H
MeSH term(s)	Benzhydryl Compounds/analysis ; Catalysis ; Coumestrol/analysis ; Endocrine Disruptors/analysis ; Estradiol/analysis ; Estrone/analysis ; Ferrous Compounds/chemistry ; Hydrogen Peroxide/chemistry ; Hydrogen-Ion Concentration ; Indoles/chemistry ; Iron/chemistry ; Manganese/chemistry ; Organometallic Compounds/chemistry ; Oxidants/chemistry ; Oxidation-Reduction ; Phenols/analysis ; Phytoestrogens/analysis ; Water Pollutants, Chemical/analysis ; Water Purification/methods
Chemical Substances	Benzhydryl Compounds ; Endocrine Disruptors ; Ferrous Compounds ; Indoles ; Organometallic Compounds ; Oxidants ; Phenols ; Phytoestrogens ; Water Pollutants, Chemical ; iron phthalocyanine ; Estrone (2DI9HA706A) ; Manganese (42Z2K6ZL8P) ; Estradiol (4TI98Z838E) ; Hydrogen Peroxide (BBX060AN9V) ; Iron (E1UOL152H7) ; bisphenol A (MLT3645I99) ; phthalocyanine (V5PUF4VLGY) ; Coumestrol (V7NW98OB34)
Language	English
Publishing date	2018-09-18
Publishing country	Germany
Document type	Journal Article
ZDB-ID	1178791-0
ISSN	1614-7499 ; 0944-1344
ISSN (online)	1614-7499
ISSN	0944-1344
DOI	10.1007/s11356-018-3215-4
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