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Issue 19, 2018
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Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase

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Abstract

Alkaline phosphatase (ALP) is an important diagnostic indicator for various human diseases including bone diseases, liver dysfunction, diabetes, breast and prostatic cancers. However, the conventional methods for ALP assay are usually cumbersome and time-consuming with low sensitivity. Here, we develop a new fluorescent method for ultrasensitive detection of ALP activity on the basis of primer dephosphorylation-initiated isothermal circular exponential amplification. We design two dual-functional hairpin probes (HP1 and HP2), which function as both the templates for exponential amplification reaction (EXPAR) and the generators for signal output. In the presence of ALP, the 3′-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3′ protruding end of HP1 to initiate the first strand displacement amplification (SDA), producing trigger 1 and fluorescence signal. The released trigger 1 is complementary to the 3′ protruding end of HP2 for the initiation of the second SDA, producing trigger 2 and fluorescence signal. Notably, trigger 2 is complementary to the 3′ protruding end of HP1 and may subsequently initiate two consecutive SDAs, enabling circular EXPAR to generate an amplified fluorescence signal. This method exhibits high sensitivity with a detection limit of 2.0 × 10−10 U μL−1 and a large dynamic range of 5 orders of magnitude from 1.0 × 10−9 to 1.0 × 10−4 U μL−1, and it can measure ALP at the single-cell level. Importantly, this method can be applied for the measurement of kinetic parameters and the screening of potential inhibitors, providing a powerful tool for ALP-related biomedical research and clinical diagnosis.

Graphical abstract: Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase

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Publication details

The article was received on 24 Aug 2018, accepted on 31 Aug 2018 and first published on 03 Sep 2018


Article type: Paper
DOI: 10.1039/C8AN01647J
Citation: Analyst, 2018,143, 4606-4613
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    Primer dephosphorylation-initiated circular exponential amplification for ultrasensitive detection of alkaline phosphatase

    L. Wang, Z. Wang and C. Zhang, Analyst, 2018, 143, 4606
    DOI: 10.1039/C8AN01647J

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