Sandwich immunoassay coupled with isothermal exponential amplification reaction: An ultrasensitive approach for determination of tumor marker MUC1

Abstract

An ultrasensitive strategy based on sandwich immunoassay coupled with isothermal exponential amplification reaction (IMEXPAR) is proposed for the determination of tumor protein Mucin 1 (MUC1). An immuno-PCR plate was prepared from modification of the primary MUC1-antibody (Ab1) onto the inner-well of the PCR plate. A biotinylated secondary MUC1-antibody tagged with the biotinylated EXPAR primer (P-Ab2) was prepared through biotin-streptavidin reaction. In the presence of target MUC1, sandwich-type combinations were specifically formed in the immuno-PCR plate. With further addition of amplification template, polymerase and nicking enzyme, EXPAR was specifically triggered, producing numerous primer replica in minutes, and greatly enhanced fluorescence of SYBR Green I. The proposed strategy has a good linear relationship with the logarithm of the MUC1 concentration ranging from 3 pM to 3 nM with a limit of detection of 1.63 pM (S/N = 3), which is two orders of magnitude lower than those of other methods. Owing to the specificity of immuno-reaction and EXPAR, the selectivity of the strategy is favorable, even if for the homologous protein. The proposed strategy was further applied for the MUC1 determination in human serum, and a satisfactory recovery range of 98.7%–105.3% was obtained. The strategy can be facilely extended to the ultrasensitive determination of various proteins.