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

Abstract

We report the use of silver hydroxylamine nanoparticles functionalised with single stranded monothiolated DNA for the detection of fungal infections. The four different species of fungi that were targeted wereCandida albicans,Candida glabrata,Candida kruseiandAspergillus fumigatus. Rational design of synthetic targets and probes was carried out by carefully analysing the 2-D folding of the DNA and then by global alignment of the sequences to ensure specificity. The effects of varying the concentrations of the DNA and dye surrounding the nanoparticles on the resultant surface enhanced Raman scattering (SERS) signal were also investigated to ensure compatibility of the probes in a multiplexed environment. Using principal components analysis (PCA) it was possible to detect the individual presence of each target and group them accordingly. The move to detect theC. kruseisingle stranded PCR product (ssPCR) was significant to demonstrate that the methodology could be employed for the detection and diagnosis of invasive fungal infections (IFDs) within a clinical setting. Initially the PCR product was subjected to an alkali shock method in order to separate the strands ready for detection using the nanoparticle probes system. This time 18 base probes were employed to enhance hybridisation efficiency and dextran sulfate was found to have a vital role in ensuring that detection of theC. kruseitarget was achieved. This demonstrated the use of DNA functionalised silver nanoparticle for the detection of clinically relevant DNA relating to a specific fungal infection and offers significant promise for future diagnostic applications.