A Competitive Enzyme-Linked Aptamer Assay(ELAA) for Determination of Tetracycline
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A competitive enzyme-linked aptamer assay for DA was performed by using two aptamers, individually; one is a 57 mer-RNA aptamer and the other is its homolog DNA aptamer. The difference between the RNA aptamer and the DNA aptamer are based on their particular nucleotides. It is known that the lack of a hydroxyl group in the 2' position of DNA is related with its chemical and biological stability. Thus, the use of the DNA homolog of the RNA aptamer could improve the affinity toward DA due to stability and finally, lower the detection limit. In this paper, we report advantageous sensitivity and specificity of its homolog DNA aptamer assay as compared to the RNA aptamer assay. Both aptamer assays were performed with 0.01 µg mL⁻¹ of each aptamer and 1.205 × 10⁻⁸ M DA-HRP conjugate using the optimized method. A dose-response curve was constructed, and the limit of detection for the DA was determined as 6.3 × 10⁻⁸ M for RNA aptamer assay, and 3.2 × 10⁻¹² M for the homolog DNA aptamer assay, respectively. These results demonstrated that the assay sensitivity was more than 10⁴ times improved with the DNA homolog of the RNA aptamer compared to its original RNA aptamer obtained through SELEX process. Also these results confirmed that the DNA homolog of the RNA aptamer can maintained the binding site and retained a function in both structure. Thus, the switching to the DNA version of RNA aptamer is possible to bind more stably and still able to bind to dopamine.
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A novel enzyme-linked aptamer assay is reported for the determination of aflatoxin B1 (AFB1). AFB1 can competitively bind with the immobilized biotin-aptamer and release biotin complementary DNA, leading to the gradual fading of the detection system color with increasing of AFB1 concentration. In the absence of AFB1, the biotinylated complementary DNA is not be released from the fixed aptamer. Therefore, the enzyme reaction occurs in the detection system. Under the optimized experimental conditions, the proposed method possessed a wide linear range for AFB1 from 1 to 80 ng/mL (R2 of 0.990) with a low detection limit of 0.36 ng/mL. The method was then applied to detect uncontaminated peanuts fortified with different concentrations of AFB1. The recovery values were from 82.60% to 94.43%, which indicated the proposed method may be used to detect AFB1 in food and has potential for the development of test kits.
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