Amplification of Molecular Recognition Signal on Imprinted Polymers Using Hybridization Chain Reaction

Abstract

Hybridization chain reaction (HCR)-based amplification strategies have shown excellent prospects of application in detection thanks to its non-enzymatic and isothermal features. For protein biomarker detection, most bioanalytical assays are dependent on antibodies or aptamers to provide specific recognition to allow single strand DNA (ssDNA) to initiate specific HCR to realize effective signal amplification. Molecularly imprinted polymers (MIPs) are excellent alternative molecular recognition materials because of low cost and high stability. In this work, we developed a non-enzymatic, HCR-amplified protein detection/ quantification using MIP as a recognition element for hemoglobin (Hb). The MIP was prepared by Pickering emulsion polymerization using mesoporous silica nanoparticles as a stabilizer, producing surface-accessible binding sites for Hb. To demonstrate specific detection of proteins, Hb captured by the MIP beads was reacted with a ssDNA to add the initiator sequence of the HCR to the captured protein in situ. After addition of two hairpin DNA molecules that were labeled with fluorophores and quenchers, the HCR reaction took place and led to the opening of the hairpins and formation of nicked dsDNA, with concomitant emission of fluorescence. A linear relationship between fluorescence intensity and the logarithm protein concentration in the range of 0.01−1 mg/mL was obtained. The limit of Hb detection was as low as 0.006 mg/mL. In addition, the non-enzymatic, isothermal fluorescent detection exhibited high selectivity, satisfactory recovery, and good repeatability. This work opens possibilities of using MIP and nucleic acid amplification to achieve low-cost, simple, and reliable detection and quantification of protein biomarkers.