Hydrogen-Bond-Engineered Photoactivated Au₁₀ Clusterzymes for Amplex Red Specificity and Highly Efficient...

Area of Science:

• Nanomaterials Science
• Biotechnology
• Analytical Chemistry

Background:

• Gold clusterzymes show promise for biosensing but are limited by reliance on hydrogen peroxide (H2O2) and acidic conditions.
• Developing H2O2- and peroxidase-independent fluorescence platforms at neutral pH is a significant challenge.

Purpose of the Study:

• To develop a highly active, substrate-specific, photoactivated oxidase-like gold (Au10) clusterzyme platform for sensitive fluorescence sensing.
• To engineer a biomimetic substrate pocket for enhanced enzyme-substrate interaction and catalytic efficiency at neutral pH.

Main Methods:

• Functionalization of Au10 clusterzymes with a thymine derivative (ATT) to create a substrate-binding pocket.
• Utilizing amplex red (AR) as a substrate for fluorescence detection.
• Photoexcitation of Au10 clusterzymes to generate ROS and electron-hole pairs for H2O2-independent oxidation of AR.

Main Results:

• The ATT-functionalized Au10 clusterzymes exhibited selective AR recruitment via hydrogen bonding, enhancing catalytic efficiency and specificity at pH 7.4.
• The platform demonstrated rapid signal saturation (5 min) and excellent kinetics (Km = 0.3201 μM, Kcat = 100.1 s−1, Kcat/Km = 3.128 × 10^8 s−1·M−1).
• Ultrasensitive quantification of acetylcholinesterase (AChE) and alkaline phosphatase (ALP) was achieved with low limits of detection (0.0125 and 0.128 mU/mL, respectively) and validated in human serum.

Conclusions:

• Hydrogen-bond microenvironment engineering of clusterzymes optimizes substrate interaction, advancing high-performance biosensing.
• The developed platform offers a robust, H2O2-independent method for sensitive enzyme detection and inhibitor screening.
• This approach overcomes limitations of traditional POD-mimicking systems, paving the way for improved diagnostic tools.