The uranyl ion(UO22+) poses high risks to human health and the environment, hence its detection and monitoring is of utmost significance. However, the development of an ultra-sensitive, high-efficiency and convenient approach for on-site detection of UO22+ remains a challenge. Herein, a reliable and reusable surface-enhanced Raman spectroscopy（SERS）-based microfluidic biosensor was developed for rapid detection of UO22+ in real samples. The detection protocol involved the reaction of 5′-Rhodamine B（RhB）-labeled double-stranded DNA for UO22+-specific DNAzyme-cleavage reaction in a U-shaped microchannel. Then, the reaction products were delivered into three parallel samples for high-throughput tests by SERS biochips,where 3 D ZnO-Ag mesoporous nanosheet arrays（MNSs） were modified with a single-stranded DNA（ssDNA）. The ssDNAwas sequence-complementary with the 5′-RhB-labeled cleaved-stranded DNA（csDNA） from the reaction products. By the hybridization of ssDNA and csDNA, the signal probe RhB was fixed close to the surface of the ZnO-Ag MNSs to enhance the Raman signal. The limit of detection for UO22+ with the microfluidic-SERS biosensor was 3.71×10-15 M. An over 20,000-fold selectivity towards UO22+ response was also achieved in the presence of 15 other metal ions. The high-throughput microfluidicSERS biosensor operated well for practical UO22+ detection, with excellent recoveries in contaminated river and tap water from95.2% to 106.3%(relative standard deviation（RSD） <6.0%, n=6). Although the SERS-based microfluidic biosensor developed in this study was deployed for the detection of UO22+, the reusable and high-efficiency system may be expanded to the detection of other analytes on-site.
