Yoctomole detection of proteins using solid phase binding-induced DNA assembly

Abstract

A binding-induced DNA assembly (BINDA) strategy is able to covert target-binding to amplifiable DNA assembly, thereby extending advances in DNA amplification to the detection of other non-amplifiable molecules. We describe here the concept of solid phase BINDA and demonstrate an approach of detecting proteins using affinity capture of the specific proteins on magnetic beads followed by BINDA. The antibody-coated magnetic beads are first introduced to capture target proteins from sample solutions. The unbound components are removed by washing. Two affinity probes, each conjugated to a DNA motif, are utilized to bind to the captured protein molecules. The binding of the two probes to the same target molecule induces assembly of the two DNA motifs that are conjugated to affinity ligands. Enzymatic ligation of the two DNA motifs generates a new DNA sequence that is then measured with real-time PCR. The highly sensitive real-time PCR detection of the DNA assembly provides an indirect detection of the target proteins that is responsible for the binding-induced DNA assembly. The incorporation of solid phase affinity capture effectively reduces the potential interferences from complex matrix and further improves the sensitivity of protein detection by pre-concentration. An application of the technique to the determination of prostate specific antigen in dilute serum shows a detection limit of 200 yoctomole (2×10(-22) mol) from a 20-μL sample. The concentration detection limit (10 attomolar) is 10-fold lower than using the homogeneous BINDA. Substantial reduction of background is a main reason for the improvement in detection limits observed with the BINDA assays. Solid phase BINDA complements to homogeneous BINDA for accomplishing ultrasensitive protein detection.

Keywords: Affinity binding; Binding-induced DNA assembly; DNA motifs; Prostate specific antigen; Proteins; Real-time PCRImmuno-PCR; Solid phase affinity capture; Ultrasensitive protein detection.