Plasmonic Single-Molecule Affinity Detection at 10^-20 Molar.

Area of Science:

• Biotechnology and Biosensing
• Surface Plasmon Resonance (SPR) Spectroscopy
• Molecular Diagnostics

Background:

• DNA amplification via replication is efficient, but comparable protein detection in immunoassays remains a challenge.
• Current Surface Plasmon Resonance (SPR) assays typically detect concentrations around 10⁻⁹ molar.
• There is a need for highly sensitive protein detection methods for advanced diagnostics.

Purpose of the Study:

• To demonstrate plasmonic single-molecule assays for both proteins and DNA.
• To achieve ultra-low limits of detection (LODs) using SPR.
• To investigate the underlying amplification mechanism for enhanced SPR performance.

Main Methods:

• Developed a millimeter-wide SPR surface functionalized with a physisorbed biolayer of recognition elements.
• Utilized acidic or alkaline pH-conditioning of the biolayer.
• Employed potentiometric and surface-probing imaging experiments to analyze the phenomenon.

Main Results:

• Achieved limits-of-detection as low as 10⁻²⁰ molar (1 ± 1 molecule in 0.1 mL) for proteins and DNA.
• Demonstrated assay performance in human serum within 1 hour.
• Observed an eleven-orders-of-magnitude improvement in SPR LODs compared to typical methods.
• Identified a pH-conditioning-induced amplification process involving self-propagating protein aggregation and electrostatic rearrangement.

Conclusions:

• The study reveals an unexplored amplification mechanism in SPR biosensing through pH-conditioned biolayers.
• This process significantly enhances sensitivity, enabling detection at the physical limits.
• The findings pave the way for highly sensitive, point-of-care SPR-based diagnostic systems.