Single-Particle Enzyme Activity Assay with Spectral-Resolved Dark-Field Optical Microscopy

Area of Science:

• Biomedical Engineering
• Nanotechnology
• Analytical Chemistry

Background:

• Enzymes are critical biomarkers for diagnosing human diseases in clinical assays.
• Accurate quantification of enzyme activity, such as alkaline phosphatase (ALP), is vital for disease detection.
• Existing methods for enzyme quantification may lack the sensitivity required for early disease diagnosis.

Purpose of the Study:

• To develop a novel spectral-resolved single-particle detection (SPD) method for quantifying alkaline phosphatase (ALP) activity.
• To utilize a supraparticle (SP) probe based on MnO2-modified gold nanoparticles (GNP@MnO2 SP) for enhanced sensitivity.
• To establish a highly sensitive assay for ALP detection in human serum for potential disease diagnosis.

Main Methods:

• A supraparticle probe (GNP@MnO2 SP) was synthesized for ALP detection.
• ALP activity was measured by observing the scattering color change of individual nanoparticles.
• Spectral-resolved dark-field optical microscopy was employed for single-particle analysis.

Main Results:

• The method achieved a linear dynamic range of 0.06 to 2.48 mU/mL (R² = 0.99) for ALP.
• A highly sensitive limit of detection of 5.8 μU/mL for ALP was demonstrated.
• The single-particle detection method proved more sensitive than ensemble measurement techniques.

Conclusions:

• The developed spectral-resolved SPD method enables ultrasensitive quantification of ALP activity.
• This approach offers a promising new strategy for designing sensitive diagnostic tools for disease biomarkers.
• The GNP@MnO2 SP probe facilitates sensitive detection via localized surface plasmon resonance (LSPR) effects.