Complete antigen-bridged DNA strand displacement amplification immuno-PCR assay for ultrasensitive detection of salbutamol

Abstract

Monitoring of low-level analytes are typical examples for analytical challenges. Salbutamol (SAL), a phenol-β2-agonist, has a very low residual content in the environment. Here, we present an ultrasensitive complete antigen-bridged PCR assay for detecting salbutamol (SAL). These DNA probes modified SAL complete antigens target recognition SAL antibodies and agglutinate synthetic DNA conjugates, thus enabling ligation of DNA probes to form a full-length DNA amplicon that contained a recognition site for cleavage endonuclease and subsequent quantification by qPCR. Moreover, SAL antibodies were modified with magnetic beads which were used to reduce the background noise and sample matrix effect, and the DNA signals were isothermally amplified by strand displacement amplification technology. Some key parameters which influence assay performance were optimized: the length of the bridge oligonucleotide, the concentration of immunomagnetic beads, SAL probes, and initiation chain, etc. Under the optimum conditions, the signal amplification of proposed Immuno-PCR assay for the detection of SAL was exponential, resulting in high potential sensitivity(~1 fg/mL) and a broad detection dynamic range (> 105 fold). Using this proposed method, we detected SAL in spiked tap water and urine samples with acceptable recoveries ranging from 88.1 to 103.3%. Theoretically, the method developed here has broad applicability and practical utility in immunoassays of a wide variety of analytes.