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

Area of Science:

• Biotechnology
• Molecular Biology
• Assay Development

Background:

• Binding-induced DNA assembly (BINDA) converts target binding into amplifiable DNA signals.
• Extending DNA amplification to non-amplifiable molecules like proteins is crucial for sensitive detection.
• Solid-phase assays offer advantages in reducing matrix interference and enabling pre-concentration.

Purpose of the Study:

• To develop and demonstrate a solid-phase BINDA strategy for ultrasensitive protein detection.
• To utilize antibody-coated magnetic beads for affinity capture of target proteins.
• To achieve highly sensitive detection of proteins through DNA assembly and real-time PCR.

Main Methods:

• Target proteins are captured on antibody-coated magnetic beads.
• Two DNA-motif-conjugated affinity probes bind to the captured protein, inducing DNA assembly.
• Enzymatic ligation creates a DNA sequence amplified and detected via real-time PCR.

Main Results:

• The solid-phase BINDA method successfully detected proteins with high sensitivity.
• Detection of prostate-specific antigen in serum reached a limit of 200 yoctomoles (10 attomolar concentration).
• This method showed a 10-fold improvement in detection limit compared to homogeneous BINDA due to reduced background.

Conclusions:

• Solid-phase BINDA is an effective strategy for ultrasensitive protein detection, complementing homogeneous BINDA.
• The approach significantly reduces background noise and matrix interference.
• This technique holds promise for various applications requiring highly sensitive protein quantification.