Highly Sensitive Homogeneous Electrochemiluminescence Biosensor for Alkaline Phosphatase Detection Based on Click...

Alkaline phosphatase (ALP) has been used as a diagnostic index of clinical diseases since its expression level is closely related to many pathological processes. In this work, a highly sensitive electrochemiluminescence (ECL) method for the determination of ALP based on a click chemistry-induced branched hybridization chain reaction (BHCR) for signal amplification and ultrafiltration technology for the separation of homogeneous amplification products is introduced. ALP can release copper ions from a Cu²⁺/PPi complex by hydrolyzing pyrophosphoric acid, which initiates click chemistry in the system. A BHCR amplification is triggered afterward by the long single-stranded DNA (ssDNA) generated by click chemistry, resulting in a three-dimensional double-stranded DNA (dsDNA) with a large molecular weight. Based on the characteristic that Ru(phen)₃²⁺ can stably insert into the groove of dsDNA, a large amount of Ru(phen)₃²⁺ is retained together with the amplified product after ultrafiltration, and therefore a significantly enhanced ECL signal can be obtained. The test results show that this method can be used for the quantitative determination of ALP ranging from 0.002 to 50 U/L, with a detection limit of 0.7 mU/L. This method has also been confirmed to have good selectivity and anti-interference, and the results of the analysis of the ALP content in the diluted serum samples are satisfactory, showing great application potential in clinical diagnosis.