Study for a Novel Diagnostic Performance of Microparticle Diatom-ZnO-APDMS Based on Fungus

Abstract

Background and purpose: The expanding spectrum and rising incidence of invasive aspergillosis (IA) makes the early-accurate identification of fungal pathogen a daunting task. Recently, as the nucleic acid-based diagnostics are widely used for clinical applications, the efficiency of DNA extraction of the fungal becomes one of the particular importance developing technologies in medical diagnostic applications. In this research, we first combined the nanotechnology for the fungal DNA extraction and developed a rapid, economical and reliable system to easily extract the genomic DNA from Aspergillus fumigatus. Our method took advantage of the diatomaceous earth (DE) to enrich the spores sample to make sure the spores can be captured even in low spores content sample. Meanwhile, the burgeoning nanomaterial ZnO performed the spores membrane breaking process, we synthesized a novel triphase micropartcles DE-ZnO-APDMS to extract the DNA from Aspergillus fumigatus. The quality and quantity of the extracted DNA were sufficient for the further diagnostics in polymerase chain reaction (PCR) amplification. So, this study confirms that the triphase micropartcles DE-ZnO-APDMS is a good candidate for varies bio-applications, especially in the diagnostic performance.

Materials and Methods: Single ZnO nanoparticles and triphase micropartcles DE-ZnO-APDMS were synthesized using the hydrothermal method in an alkaline medium. The morphology of DE, ZnO, and DE-ZnO-APDMS was characterized using field-emission scanning electron microscopy (FE-SEM) on a JSM-7500F instrument (JEOL) to confirm the DE and ZnO structure as well as the decoration of ZnO on DE. Aspergillus fumigatus which is the most common species cause invasive aspergillosis was used in all experiments in this study. In order to confirm the DE-APDMS can enrich the Aspergillus fumigatus spores in large Sample volume, the traditional fungal culture method was used. Meanwhile, the DNA binding ability of DE-APDMS and DE-ZnO-APDMS were tested by the adsorption equilibrium experiment with UV-Vis spectroscopy. The Aspergillus fumigatus DNA extracted by ZnO and DE-ZnO-APDMS were compared with the DNA extraction from commercial kit. The quality and quantity of the extracted DNA were sufficient for the further diagnostics in polymerase chain reaction (PCR) amplification.

Results: The firstly synthesized triphase micropartcles DE-ZnO-APDMS showed that the multigonal star-shape nanoparticles ZnO (~300nm) grew on the microscale hollow silica structures which have lots of nanoscale pores in the wall. The DE-APDMS can enrich the limited amount Aspergillus fumigatus spores in large sample volume. Both The DE-APDMS and DE-ZnO-APDMS had the DNA binding ability because the DNA form the spores was cross-linked by the composite surface modifier APDMS. The synthesized nanoparticles ZnO (~300nm) perform the successful lysis of fungal spores. The unique triphase micropartcles DE-ZnO-APDMS combined the advantages of the each part in the composite, it can extract fungal DNA in this all-in-one assay. Compared with the commercial kit assay, the fungal DNA isolation assay based on the triphase micropartcles DE-ZnO-APDMS was more sensitive in large sample volume with limited numbers of spores.

Conclusions: In this study, a simple and rapid fungal DNA isolation assay based on synthesized triphase micropartcles DE-ZnO-APDMS for the diagnosis of invasive aspergillosis is presented. With quantitative PCR analysis of the amplified DNA isolated using the DE-ZnO-APDMS-based fungal DNA isolation assay and commercial kit assay, our results show that these uniquely synthesized triphase micropartcles DE-ZnO-APDMS was more effective to extract the fungal DNA from samples with less density. This study introduces a new candidate for diagnostic techniques for human invasive aspergillosis.