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Selection, Characterization, and Application of ssDNA Aptamer against Furaneol.

ABSTRACT: Furaneol is an aroma compound which occurs naturally in foods and is used as an artificial flavor. Detection of furaneol is required in food science and food processing industry. Capture- Systematic Evolution of Ligands by EXponential enrichment (SELEX) protocol was applied for the isolation of an aptamer binding to furaneol, a small volatile organic substance contributing to the flavor of various products. Thirteen cycles of selection were performed. The resulting DNA pool was cloned, using blunt-end cloning, and ninety-six plasmids were sequenced and analyzed. Eight oligonucleotides were selected as aptamer candidates and screened for the ability to bind to furaneol, using three different methods-magnetic-beads associated elution assay, SYBR Green I assay, and exonuclease protection assay. One of the candidates was further characterized as an aptamer. The apparent equilibrium constant was determined to be (1.1 ± 0.4) µM, by the fluorescent method. The reported aptamer was applied for development of the ion-sensitive field-effect transistor (ISFET)-based biosensor, for the analysis of furaneol, in the concentration range of 0.1⁻10 µM.

SUBMITTER: Komarova N

PROVIDER: S-EPMC6320952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI). However, the current program for NGAL detection is not extensively applied in clinics due to the high expense of antibodies. Nucleic acid aptamers are single-strand DNAs or RNAs which could bind to targets with high specificity and affinity, and they have been widely used in the diagnosis and therapy for multiple diseases. It is valuable for us to develop a new method for NGAL detection using aptamers instead of antibodies to achieve increased efficiency and decreased cost.MethodsNucleic acid aptamers against NGAL were obtained after SELEX process using magnetic beads, and an enzyme-linked aptamer analysis (ELAA), which can be widely used in clinical diagnosis at low cost, were successfully established. The feasibility of ELAA was further validated with urine samples harvested from 43 AKI patients and 30 healthy people.ResultsThree candidate aptamers, including NA36, NA42 and NA53, were obtained after 8 rounds of SELEX process with magnetic beads and verified by quantitative polymerase chain reaction (qPCR), and the Kd value of each aptamer was 43.59, 66.55 and 32.52 nM, respectively. Moreover, the linear relationship was consistent at the range of 125-4000 ng/mL, and the detection limit of ELAA assay was 30.45 ng/mL. We also found that NGAL could be exclusively detected with NA53, and no cross-reaction between NA53 and human albumin or globulin occurred, the coefficient of variation (CV) between inner-plate and inter-plate was less than 15%, and the recovery rate was between 80 and 110%. Moreover, the sensitivity and specificity of ELAA assay in this study are 100% and 90%, respectively. Consistently, these results could also diagnose whether the occurrence of AKI in lots of patients, which has been demonstrated with the ELAA method we established after using NA53.ConclusionsTaken together, NA53, the best candidate aptamer targeting NGAL protein, can be applied in clinical testing.

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Selection, Characterization, and Application of ssDNA Aptamer against Furaneol.

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