OBJECTIVE: To develop a novel colorimetric method for detecting the tumor biomarker vascular endothelial growth factor (VEGF) based on aptamer and magnetic beads. METHODS: The capture aptamer was hybridized to urease functionalized single-stranded DNA (ssDNA) and immobilize on the surface of magnetic beads by specific biotin-avidin binding. In the presence of VEGF, aptamers bound to VEGF to form a specific stem-loop structure to release the urease functionalized ssDNA. After separation, the supernatant was transferred to a tube and urea and phenol red were added. Urease hydrolyzed urea to produce ammonia to cause an increase of the pH value and a color change of phenol red. The results were inspected with either the naked eyes or by a UV spectrophotometer. RESULTS: Under optimized conditions, the detection system showed a good linear relationship for VEGF detection in the range of 0.1 to 10 pmol/L with a detection limit as low as 0.06 pmol/L. The results of VEGF detection in the serum of patients with lung cancer were consistent with those using an ELISA Kit. The results of examination of 10 serum samples with this aptamer-based method and ELISA kit showed that the accuracy of this method was 90%. CONCLUSION: This aptamer-based system provides an simple and convenient method for VEGF detection with a high sensitivity and selectivity.
OBJECTIVE: To develop a novel colorimetric method for detecting the tumor biomarker vascular endothelial growth factor (VEGF) based on aptamer and magnetic beads. METHODS: The capture aptamer was hybridized to urease functionalized single-stranded DNA (ssDNA) and immobilize on the surface of magnetic beads by specific biotin-avidin binding. In the presence of VEGF, aptamers bound to VEGF to form a specific stem-loop structure to release the urease functionalized ssDNA. After separation, the supernatant was transferred to a tube and urea and phenol red were added. Urease hydrolyzed urea to produce ammonia to cause an increase of the pH value and a color change of phenol red. The results were inspected with either the naked eyes or by a UV spectrophotometer. RESULTS: Under optimized conditions, the detection system showed a good linear relationship for VEGF detection in the range of 0.1 to 10 pmol/L with a detection limit as low as 0.06 pmol/L. The results of VEGF detection in the serum of patients with lung cancer were consistent with those using an ELISA Kit. The results of examination of 10 serum samples with this aptamer-based method and ELISA kit showed that the accuracy of this method was 90%. CONCLUSION: This aptamer-based system provides an simple and convenient method for VEGF detection with a high sensitivity and selectivity.
Authors: David F Thompson; William Eborall; Andrew Dinsmore; Christopher J Smith; Catherine J Duckett Journal: Rapid Commun Mass Spectrom Date: 2010-04-15 Impact factor: 2.419