DNA encapsulating liposome based rolling circle amplification immunoassay as a versatile platform for ultrasensitive detection of protein.

ABSTRACT

A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. A biobarcode design was incorporated in the technique, which allowed the strategy to be directly implemented for multiplex assay of multiple proteins. Also, the technique allowed easy preparation of the DNA-carrying antibody reagent and the implementation with simple instrumentation. The technique was demonstrated for the determination of prostate-specific antigen (PSA), a highly selective biomarker associated with prostate cancer. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg mL(-1) to 0.1 ng mL(-1) with a limit of detection as low as 0.08 fg mL(-1) and a high dose-response sensitivity. The liposome-RCA immunoassay holds great promise as a versatile, sensitive, and robust platform to combine the nucleic acid amplification with immunoassay for ultrasensitive protein detection.