scholarly journals Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 134 ◽  
Author(s):  
Marc Prante ◽  
Torsten Schüling ◽  
Bernhard Roth ◽  
Kort Bremer ◽  
Johanna Walter
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Detection Methods ◽  
Microarray Experiments ◽  
Hydroxyvitamin D ◽  
Detection Approach ◽  
Biosensor System ◽  
25 Hydroxyvitamin D ◽  
Current Detection

Detection of the small molecule 25-hydroxyvitamin D (25(OH)D) as the most relevant marker for vitamin D supply suffers from a high variability of results using the current detection methods, such as high-performance liquid chromatography (HPLC) and immunoassays. A new detection approach using a highly specific aptamer directed against 25(OH)D was established in this study based on the target-induced dissociation (TID) sensing approach. In this work, the aptamer was investigated regarding its structural properties as well as its binding affinity by using microscale thermophoresis (MST). Moreover, complementary oligonucleotides were designed based on the aptamer structure and were evaluated in MST experiments. Binding experiments of immobilized aptamers were conducted in microarray experiments. It could be shown that the aptamer exhibited the usual B-DNA structure and did not form any G-quadruplexes. The design of complementary oligonucleotides for the TID assay identified a putative 25(OH)D binding site within the aptamer. The limit of detection of the established competitive assay was determined to be 5.4 nM, which sets the stage for the development of a biosensor system.

scholarly journals Graphene Oxide and Fluorescent Aptamer Based Novel Biosensor for Detection of 25-hydroxyvitamin D3

2021 ◽  
Author(s):  
Ritika Gupta ◽  
Sunaina Kaul ◽  
Vishal Singh ◽  
Sandeep Kumar ◽  
Nitin Kumar Singhal
Keyword(s):  
Vitamin D ◽  
Graphene Oxide ◽  
Limit Of Detection ◽  
Low Cost ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Detection Methods ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Sensitive Sensor

Abstract For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5’, 6-FAM) is adsorbed by π -π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.

scholarly journals Graphene oxide and fluorescent aptamer based novel biosensor for detection of 25-hydroxyvitamin D3

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ritika Gupta ◽  
Sunaina Kaul ◽  
Vishal Singh ◽  
Sandeep Kumar ◽  
Nitin Kumar Singhal
Keyword(s):  
Vitamin D ◽  
Graphene Oxide ◽  
Limit Of Detection ◽  
Low Cost ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Detection Methods ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Sensitive Sensor

AbstractFor maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D3 (25(OH)D3). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D3 levels in our blood. Quantification techniques for 25(OH)D3 are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D3 was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5’, 6-FAM) is adsorbed by π–π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D3, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D3. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D3 by this method was found to be 0.15 µg/mL whereas when GO-COOH was used, limit of detection was improved to 0.075 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.

Analysis of Chloramphenicol and Two Metabolites in Crab and Shrimp Following Waterborne Exposure

2018 ◽  
Vol 81 (4) ◽  
pp. 677-683
Author(s):  
EDWARD L. E. JESTER ◽  
JARED I. LOADER ◽  
HAROLD A. FLORES QUINTANA ◽  
KATHLEEN R. EL SAID ◽  
RONALD A. BENNER ◽  
...  
Keyword(s):  
United States ◽  
Limit Of Detection ◽  
Parent Drug ◽  
The United States ◽  
Detection Methods ◽  
Health Issues ◽  
Drug Molecule ◽  
Waterborne Exposure ◽  
Aquaculture Products ◽  
Current Detection

ABSTRACT The use of chloramphenicol (CAP) in aquaculture products is banned in many countries, including the United States, due to human health issues. Very few depletion and metabolism studies of CAP in seafood have been performed. Current detection methods for CAP residues in food are directed toward the parent drug molecule, but rapid elimination following treatment suggests the need for an alternative marker residue. We identified, characterized, and determined the persistence of two CAP metabolites, CAP-base (CAP-B) and CAP-alcohol (CAP-OH), in crab and shrimp. Interday recoveries of CAP, CAP-B, and CAP-OH in muscle fortified (n = 9) at levels of 0.15 to 0.60 ng/g ranged from 95 to 127% and 101 to 119% for crab and shrimp, respectively, with repeatability ranging from 4 to 19%. The limit of detection for CAP and metabolites in crab and shrimp ranged from 0.05 to 0.11 ng/g. We also monitored the depletion of CAP, CAP-B, and CAP-OH in crab following waterborne exposures. To our knowledge, we present the first CAP depletion and metabolite study following waterborne exposure in crabs, with the aim of identifying alternative marker residues.

scholarly journals Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection

2021 ◽  
Author(s):  
Xiangxiang Zhao ◽  
Zhengduo Wang ◽  
Bowen Yang ◽  
Zilong Li ◽  
Yaojun Tong ◽  
...  
Keyword(s):  
De Novo ◽  
Limit Of Detection ◽  
Antigen Detection ◽  
Detection Methods ◽  
Serum Samples ◽  
Medical Diagnoses ◽  
Nucleic Acid Analysis ◽  
Amplification Strategy ◽  
Strategy Integration ◽  
Current Detection

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/μL) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 minutes. Given our system's simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

Simultaneous determination of 25-hydroxy- and 1,25-dihydroxyvitamin D from a single sample by dual-cartridge extraction.

1984 ◽  
Vol 30 (1) ◽  
pp. 56-61 ◽  
Author(s):  
P C Kao ◽  
D W Heser
Keyword(s):  
High Performance ◽  
Vitamin D Metabolites ◽  
Hydroxyvitamin D ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Hydroxylated Metabolites ◽  
Silica Cartridge ◽  
25 Hydroxyvitamin D ◽  
The Mean

Abstract With this dual-cartridge system we extract 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] from a single serum sample by using a nonpolar octadecylsilanol silica cartridge to adsorb the vitamin D metabolites and other nonpolar substances; the polar substances wash through the cartridge. The eluted material is then applied to a second alkylamine cartridge, which adsorbs the relatively polar hydroxylated metabolites; the less-polar substances are washed from the second cartridge. Elution from the second cartridge purifies and also separates 25(OH)D and 1,25(OH)2D with analytical recoveries near 90%. The monohydroxyl metabolites are determined by "high-performance" liquid chromatography (HPLC); the dihydroxyl metabolites are further purified by HPLC and determined by radioreceptor assay according to established procedures. Mean (+/- SD) winter normal values (34 subjects of both sexes; blood drawn in mid-April) were 18 +/- 5 micrograms/L for 25(OH)D and 25 +/- 7 ng/L for 1,25(OH)2D. In nine laboratory volunteers, the mean increase in the serum 25(OH)D3 value 5 h after ingestion of 50 micrograms of 25-hydroxycholecalciferol (Calderol) was 9 (SD 4) micrograms/L.

Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography.

1978 ◽  
Vol 24 (2) ◽  
pp. 287-298 ◽  
Author(s):  
G Jones
Keyword(s):  
Vitamin D ◽  
Protein Binding ◽  
High Performance ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Vitamin D Metabolites ◽  
Binding Assays ◽  
Hydroxyvitamin D ◽  
Chemical Assay ◽  
25 Hydroxyvitamin D

Abstract I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.

scholarly journals A Rapid and Sensitive Fluorescent Microsphere-Based Lateral Flow Immunoassay for Determination of Aflatoxin B1 in Distillers’ Grains

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2109
Author(s):  
Zifei Wang ◽  
Pengjie Luo ◽  
Baodong Zheng
Keyword(s):  
Aflatoxin B1 ◽  
High Performance ◽  
Animal Feed ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Distillers Grains ◽  
Detection Methods ◽  
Lateral Flow Immunoassay ◽  
Quantitative Detection ◽  
Fluorescent Microsphere

Aflatoxin B1 (AFB1) is a toxic compound naturally produced by the genera Aspergillus. Distillers’ grains can be used as animal feed since they have high content of crude protein and other nutrients. However, they are easily contaminated by mycotoxins, and currently there are no rapid detection methods for AFB1 in distillers’ grains. In this study, a lateral flow immunoassay (LFIA) based on red fluorescent microsphere (FM), is developed for quantitative detection of AFB1 in distillers’ grains. The whole test can be completed within 15 min, with the cut-off value being 25.0 μg/kg, and the quantitative limit of detection (qLOD) being 3.4 μg/kg. This method represents satisfactory recoveries of 95.2–113.0%, and the coefficients of variation (CVs) are less than 7.0%. Furthermore, this technique is successfully used to analyze AFB1 in real samples, and the results indicates good consistency with that of high-performance liquid chromatography (HPLC). The correlation coefficient is found to be greater than 0.99. The proposed test strip facilitates on-site, cost-effective, and sensitive monitoring of AFB1 in distillers’ grains.

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