From synthetic DNA to PCR product: detection of fungal infections using SERS

Area of Science:

• Nanotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Fungal infections pose a significant threat, necessitating rapid and accurate diagnostic methods.
• Current diagnostic techniques can be slow and lack specificity, delaying treatment for invasive fungal infections (IFIs).
• Developing sensitive and specific biosensors for fungal DNA detection is crucial for timely clinical intervention.

Purpose of the Study:

• To develop and validate a novel diagnostic approach using DNA-functionalized silver nanoparticles for detecting specific fungal species.
• To optimize nanoparticle probe design and detection parameters for enhanced sensitivity and specificity.
• To demonstrate the potential of this methodology for the clinical diagnosis of invasive fungal infections.

Main Methods:

• Synthesis and functionalization of silver nanoparticles with single-stranded DNA probes.
• Design of DNA targets and probes based on sequence analysis and secondary structure.
• Optimization of nanoparticle-DNA-dye concentrations for surface-enhanced Raman scattering (SERS) detection.
• Application of principal component analysis (PCA) for signal discrimination and identification of fungal species.
• Detection of single-stranded PCR products (ssPCR) from Candida krusei using optimized probes and dextran sulfate.

Main Results:

• The DNA-functionalized silver nanoparticles successfully detected four fungal species: Candida albicans, Candida glabrata, Candida krusei, and Aspergillus fumigatus.
• Optimization studies identified suitable DNA and dye concentrations for multiplexed SERS detection.
• PCA enabled the accurate individual detection and grouping of target fungal species.
• The method was successfully adapted to detect a specific PCR product from Candida krusei, demonstrating clinical relevance.
• Dextran sulfate was found to be critical for efficient detection of the Candida krusei target.

Conclusions:

• DNA-functionalized silver nanoparticles offer a sensitive and specific platform for detecting fungal infections.
• The developed SERS-based method shows significant promise for the rapid diagnosis of invasive fungal infections in clinical settings.
• This nanotechnology-based approach provides a foundation for future advancements in molecular diagnostics for mycoses.