Alkaline phosphatase-catalyzed silver deposition for electrochemical detection

Area of Science:

• Biochemistry
• Electrochemistry
• Biosensor Technology

Background:

• Alkaline phosphatase (AP) is a widely used enzymatic label in various bioassays like ELISAs and immunosensors.
• AP catalyzes substrate dephosphorylation, yielding a detectable product quantified via optical or electrochemical methods.
• Current detection methods can be limited in sensitivity for specific applications.

Purpose of the Study:

• To develop a highly sensitive detection method utilizing alkaline phosphatase as an enzymatic label.
• To apply this method for the detection of specific DNA sequences from pathogenic bacteria.
• To compare the sensitivity of the new method with existing techniques.

Main Methods:

• A novel substrate (3-indoxyl phosphate) was employed, which upon dephosphorylation by AP, generates a compound reducing silver ions to metallic silver.
• The localized silver deposit was electrochemically stripped and quantified using anodic stripping voltammetry.
• The method was applied to an enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes for detecting the autolysin gene of Streptococcus pneumoniae.

Main Results:

• The enzymatic deposition of silver allowed for localized signal generation at the site of AP activity.
• Anodic stripping voltammetry of the deposited silver provided a sensitive electrochemical readout.
• The developed genosensor demonstrated a 14-fold increase in sensitivity compared to direct voltammetric detection of indigo carmine.

Conclusions:

• The developed AP-based silver deposition and anodic stripping voltammetry method offers significantly enhanced sensitivity for biosensing applications.
• This approach is effective for the specific detection of bacterial virulence genes, such as the autolysin gene in Streptococcus pneumoniae.
• The method holds promise for sensitive and specific pathogen detection in clinical and environmental diagnostics.