title: An enzyme substrate binding aptamer complex based time-resolved fluorescence sensor for the adenosine deaminase detection.
abstract: In this work, we report an enzyme substrate binding aptamer complex based fluorescence sensor for an enzyme activity detection of adenosine deaminase (ADA). The sensor employs a DNA probe containing an adenosine aptamer region dually labeled with biotin and digoxigenin (DIG). The probe is immobilized in a streptavidin-modified 96-well micro plate via biotin-avidin bridge, and the DIG serves as an affinity tag for an Anti-DIG antibody conjugated with horseradish peroxidase (anti-DIG-HRP). The principle of our sensor is as follows: the aptamer forms a coiled structure making the DNA in a "closed" state in the presence of adenosine, which shields the DIG tag from the bulky anti-DIG-HRP due to a proper steric effect. After adding ADA in the test solution, adenosine will be converted to inosine regardless of the aptamer binding. Then, the inosine release causes the DNA to relax and consequently, the DIG becomes accessible to the bulky anti-DIG-HRP which will further conjugate a Eu³⁺ labeled anti-horseradish peroxidase (Eu-anti-HRP). The Eu-anti-HRP can give a fluorescence signal when an enhancement solution is added. In the result of the experiment, we found the sensor signal can reflect the enzyme activity accurately and the detection limit is lowered to 0.5 U L⁻¹ of ADA not only in buffer solution, but also in serum, and an enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride is studied. With a concentration of 0.01 nM it is enough to cause a distinct difference of the sensor response.
authors.affiliation: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Qianlu
authors.initials: Q
authors.lastname: Yang
authors.forename: Jue
authors.initials: J
authors.lastname: Zhang
authors.forename: Lixin
authors.initials: L
authors.lastname: Fu
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Bing
authors.initials: B
authors.lastname: Wu
authors.forename: Minhao
authors.initials: M
authors.lastname: Xie
authors.forename: Biao
authors.initials: B
authors.lastname: Huang
mesh_terms: D000241:Adenosine / Q000737:chemistry*; D000243:Adenosine Deaminase / Q000737:chemistry / Q000302:isolation & purification*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques; D005453:Fluorescence; D057230:Limit of Detection; D013379:Substrate Specificity
chemical_list: D052157:Aptamers, Nucleotide; D000243:Adenosine Deaminase; D000241:Adenosine