Magnetic separation and detection of a cellulase gene using core–shell nanoparticle probes towards a Q-PCR assay

Abstract

A magnetic separation and detection method for a target sequence of a gene encoding cellulase using biocompatible core–shell nanoparticle probes was developed. An aminated capture probe was conjugated with biocompatible Fe₃O₄–SiO₂–Au core–shell nanoparticles. The target probe and signal probe were hybridized with the capture probe on the surface of the inorganic DNA carrier, which resulted in core–shell nanoparticle probes. In the presence of an external magnetic field, it is convenient and time-saving to realize the detection of the cellulase gene in Trichoderma reesei (T. reesei) by liquid fermentation and subsequent magnetic separation. Quantitative PCR (Q-PCR) was performed to give absolute quantification of the concentration of the target nucleic acid, and the Q-PCR result was compared to that of the electrochemical method. The optimized experimental conditions were studied to maximize the hybridization efficiency and detection sensitivity. The amperometric current response was linearly related to the common logarithm of the target nucleic acid concentration in the range of 1.0 × 10⁻¹³ to 1.0 × 10⁻⁹ M, with a detection limit of 1.2 × 10⁻¹⁴ M.