L-Argininamide biosensor based on S1 nuclease hydrolysis signal amplification

Abstract

A sensitive method is presented for the detection of L-argininamide. It is based on the amplification of the hydrolysis of S1 nuclease of single-stranded regions of an aptamer-target complex. The S1 nuclease, which is sequence-independent, is used to “recycle” target molecules, thus leading to strongly enhanced chemiluminescence (CL). L-Argininamide was chosen as model analyte. The DNA aptamer and its complementary DNA were labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The DNA complementary to the aptamer was labeled with ABEI and immobilized on magnetic beads (MBs) coated with gold. The aptamer was also labeled with ABEI and self-assembled on the MBs. A duplex was formed due to hybridization between the DNA aptamer and the DNA complementary to the aptamer. In the presence of the target L-argininamide, a stem-loop aptamer structure is formed which subsequently denatures the duplex. This switch from a duplex structure to a stem-loop structure causes the formation of single-stranded regions both in the target-aptamer and in the single-stranded DNA on the MBs. The nuclease hydrolyzes the single-stranded regions and single-stranded DNA. Ultimately, L-argininamide is released which then interacts with another aptamer on the MB, thereby leading to one more L-argininamide. This autocatalytic cycle can generate substantial quantities of ABEI which then can be sensitively determined by the diperiodatonickelate-isoniazide reaction system. L-argininamide can be detected in the concentration range from 3.0 × 10−4 to 3.0 × 10−7 M, and the limit of detection is 1.0 × 10−7 M.