Quantitative image analysis for sensing HIV DNAs based on NaGdF4:Yb,Er@NaYF4 upconversion luminescent probe and magnetic beads

A quantitative image analysis and separation system was designed for detection of human immunodeficiency virus (HIV) related DNAs in the red region by combining of upconversion luminescence nanoprobe and magnetic beads (MBs). Carboxyl-modified NaGdF4:Yb,[email protected]4 upconversion nanoparticles (UCNPs) were connected to single-stranded DNA to form a reporter probe, while carboxyl-modified magnetic beads were connected to another single-stranded DNA to form a capture probe. In the presence of target HIV DNAs, NaGdF4:Yb,[email protected]4 UCNPs and magnetic beads were conjugated to form UCNPs-DNA-MBs nanocomplexes through DNA-DNA heteroduplexes based on the principle of complementary base pairing. After magnetic separation, the upconversion luminescence intensity of UCNPs-DNA-MBs nanocomplexes was related to the concentration of the target HIV DNAs. The detection methods were investigated by fluorescence spectrometry and quantitative image analysis based on upconversion luminescence total internal reflection (TIR) imaging platform. Compared to the luminescence spectral analysis, the quantitative image analysis shows higher sensitivity (linear range of 0.02−30 nM and detection limit of 0.0102 nM). More importantly, this method only needs 10 μL sample and the analysis time is about 0.1 s. The method combines quantitative image analysis with magnetic separation technology, which has the characteristics of low background interference and high separation efficiency allowing for application to the field of biology.