A new strategy based on aptasensor to time-resolved fluorescence assay for adenosine deaminase activity.

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and medical diagnosis. The major obstacles are the background signals from the biological fluids and sensors themselves. Because the europium ion (Eu (III)) has the much longer fluorescence lifetime (1 ms) than that of the background (5 ns), time-resolved method can be widely used to eliminate the biological background. So, we report here an aptamer-based sensor (aptasensor) for time-resolved fluorescence assay of adenosine deaminase (ADA). This aptasensor employs two oligonucleotides labeled with DIG and biotin, respectively. The DNA1 (an oligonucleotide modified with biotin) is immobilized at a streptavidin-modified plate surface via the biotin-avidin bridge, and the DIG which is modified on the DNA2 serves as an affinity tag for the Eu(3+) labeled anti-DIG (Eu-anti-DIG) binding. If the adenosine is binding with DNA1, it will make the DNA1 in the closed state with a close-packed tight structure, which forbids the DNA2 approaching. And if the ADA is added into the mixture, the DNA1 unbends, because of the adenosine is transformed to inosine catalyzed by the ADA. Then DNA2 could hybridize with DNA1. Accordingly, the DIG finds Eu-anti-DIG and the Eu-anti-DIG will give a remarkable fluorescent signal. The detection limit of the aptasensor can be lowered to 2 UL(-1), which can meet the clinical requirement of ADA cutoff value (4 UL(-1)). Moreover, we were able to detect ADA in human serum quantitatively. Combined with time-resolved based measurements and aptasensor, this strategy holds great potential in protein analysis.