Abstract
A fluorescence strategy for alkaline phosphatase (ALP) assay in complicated samples with high sensitivity and strong stability is developed based on an allosteric probe (AP). This probe consists of two DNA strands, a streptavidin (SA) aptamer labeled by fluorophore and its totally complementary DNA (cDNA) with a phosphate group on the 5′ end. Upon ALP introduction, the phosphate group on the cDNA is hydrolyzed, leaving the unhydrolyzed cDNA sequence for lambda exonuclease (λ exo) digestion and releasing SA aptamer for binding to SA beads, which results in fluorescence enhancement of SA beads that can be detected by flow cytometry or microscopy. We have achieved a detection limit of 0.012 U/mL with a detection range of 0.02~0.15 U/mL in buffer and human serum. These figures of merit are better than or comparable to those of other methods. Because the fluorescence signal is localized on the beads, they can be separated to remove fluorescence background from complicated biological systems. Notably, the new strategy not only applies to ALP detection with simple design, easy operation, high sensitivity, and good compatibility in complex solution, but also can be utilized in ALP-linked immunosorbent assays for the detection of a wide range of targets.
Highlights
Alkaline phosphatase (ALP), a type of hydrolase enzyme, is widespread in nature for removing phosphate groups from various biomolecules like nucleic acids, proteins, and carbohydrates
The allosteric probe (AP) contains two complementary sequences, an SA aptamer labeled by fluorophore and its total complementary DNA (cDNA) modified with a 5’-phosphoryl terminus
An AP is composed of two complementary sequences, including an SA aptamer labeled by fluorophore and its total cDNA modified with a 5’-phosphoryl terminus
Summary
Alkaline phosphatase (ALP), a type of hydrolase enzyme, is widespread in nature for removing phosphate groups from various biomolecules like nucleic acids, proteins, and carbohydrates. Due to ALP’s important role in metabolism in the liver and development of the skeleton, an abnormal level of ALP is often related to bone disease (Garnero and Delmas, 1993), liver dysfunction (Ooi et al, 2007), diabetes (Berberoglu et al, 2007), and breast cancer (Brar et al, 1993). Development of ALP detection methods is important for clinical diagnosis and cancer screening. Like horseradish peroxidase (HRP), ALP can conjugate to streptavidin (Zhou et al, 2016) or a detection antibody for signal amplification and output in enzyme-linked immunosorbent assay (ELISA) (Porstmann et al, 1985).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
