摘要
A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deaminase assays with high sensitivity using water-soluble cationic conjugated polymers(CCPs).The assay contains three elements:a biotin-labeled aptamer of adenosine(biotin-aptamer),a signaling probe single-stranded DNA-tagged fluorescein at terminus(ssDNA-Fl) and a CCP.The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-Fl unhybridized,and the ssDNA-Fl is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field.In this case,after the addition of CCP to the magnetic beads solution,the fluo-rescence resonance energy transfer(FRET) from CCP to fluorescein is inefficient.Upon adding adenosine deaminase,the adenosine is converted into inosine,and the biotin-aptamer is hybridized with ssDNA-Fl to form doubled stranded DNA(biotin-dsDNA-Fl).The ssDNA-Fl is attached to the mag-netic beads at the separation step,and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein.Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions.The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.
A magnetically assisted fluorescence ratiometric technique has been developed for adenosine deaminase assays with high sensitivity using water-soluble cationic conjugated polymers （CCPs）. The assay contains three elements： a biotin-labeled aptamer of adenosine （biotin-aptamer）, a signaling probe single-stranded DNA-tagged fluorescein at terminus （ssDNA-FI） and a CCP. The specific binding of adenosine to biotin-aptamer makes biotin-aptamer and ssDNA-FI unhybridized, and the ssDNA-FI is washed out after streptavidin-coated magnetic beads are added and separated from the assay solution under magnetic field. In this case, after the addition of CCP to the magnetic beads solution, the fluo- rescence resonance energy transfer （FRET） from CCP to fluorescein is inefficient. Upon adding adenosine deaminase, the adenosine is converted into inosine, and the biotin-aptamer is hybridized with ssDNA-FI to form doubled stranded DNA （biotin-dsDNA-FI）. The ssDNA-FI is attached to the mag- netic beads at the separation step, and the addition of CCP to the magnetic beads solution leads to efficient FRET from CCP to fluorescein. Thus the adenosine deaminase activity can be monitored by fluorescence spectra in view of the intensity decrease of CCP emission or the increase of fluorescein emission in aqueous solutions. The assay integrates surface-functionalized magnetic particles with significant amplification of detection signal of water-soluble cationic conjugated polymers.
基金
Supported by the "100 Talents" Program of the Chinese Academy of Sciences
the National Natural Science Foundation of China (Grant No.20574073)
