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An indirect aptamer-based SERS assay for insulin-like growth factor 2 receptor (IGF-IIR) protein was developed. The gold substrate and silver nanoparticles (AgNPs) were employed simultaneously to achieve double enhancement for SERS signals. Firstly, the five commercial SERS substrates including Enspectr, Ocean-Au, Ocean-AG, Ocean-SP and Q-SERS substrates were evaluated using 4-mercaptobenzoic acid (4-MBA). The Q-SERS substrate was selected based on low relative standard deviation (RSD, 8.6%) and high enhancement factor (EF, 8.7*105), using a 785 nm laser. The aptamer for IGF-IIR protein was designed to include two sequences: one grafted on gold substrate to specifically capture the IGF-IIR protein and a second one forming a 3′ sticky bridge to capture SERS nanotags. The SERS nanotag was composed by AgNPs (20 nm), 4-MBA and DNA probes that can hybridize with the aptamer. Due to the steric-hindrance effect, when the aptamer doesn't combine with IGF-IIR protein, it only can capture the SERS nanotags. Therefore, there was a negative correlation between the concentration of IGF-IIR protein and the intensity of 4-MBA at 1076 cm−1. The detection limit reached to 141.2 fM and linear range was from 10 pM to 1 μM. The SERS aptasensor also exhibits a high reproducibility with an average RSD of 4.5%. The interference test was conducted with other four proteins to verify the accuracy of measuring. The study provides an approach to quantitative determination of proteins based on specific recognition and nucleic acid hybridization of aptamers, to establish sandwich structure for SERS enhancement. [ABSTRACT FROM AUTHOR]