یک آزمایش ساده، حساس و بدون برچسب برای فعالیت آلکالن فسفاتاز بر اساس تقویت جابجایی رشته های نمایشی هدف

We herein describe a simple, sensitive, and label-free strategy for alkaline phosphatase (ALP) activity based on target-promoted exponential strand displacement amplification (eSDA). The developed system utilizes 3′-phosphorylated DNA probes whose phosphate group at the 3′-end blocks the extension by DNA polymerase. Only in the presence of ALP, the phosphate group at the 3′-end is transformed to the hydroxyl group, which enables the extension by DNA polymerase and produces the double-stranded recognition sequence for nicking endonuclease. Consequently, the effective eSDA is executed, generating a large number of duplex products with the high fluorescence signal from SYBR green I. Based on this novel strategy, we determined the ALP activity down to 0.47 U/L with the high selectivity. In addition, the practical applicability of this assaying system was successfully demonstrated by reliably determining the ALP activity in human serum.