Photothermal mediated rolling circle amplification toward specific and direct in situ mRNA detection

Abstract

Rolling circle amplification (RCA) had the prospect of assisting clinic diagnosis with advantage in in situ mRNA detection at single cell level. However, for direct mRNA detection, RCA had relatively low detection specificity and efficiency. Here, we introduced 4-(10, 15, 20-Triphenylporphyrin-5-yl)phenylamine (TPP) modified Au nanoparticle (Au-TPP) to improve the specificity of in-situ RCA. Through photothermal effect, Au-TPP acted as the specific heat source upon irradiation of 635 nm laser. The photothermal mediated RCA would be initiated only when the Au-TPP as well as the padlock anchored adjacently on the same target mRNA. Furthermore, we introduced 'C' form target-specific oligonucleotide linker probes to make generic padlock and Au-TPP for different mRNA targets, so that for a new mRNA target one does not have to redesign the padlock and the Au-TPP probe. By these strategies, we successfully developed a specific and photothermal mediated hyperbranched rolling circle amplification for direct in situ mRNA detection, suitable for both formalin-fixed paraffin-embedded (FFPE) tissue section and frozen tissue section.

Keywords: Formalin-fixed paraffin-embedded tissue section; Frozen tissue section; In situ mRNA detection; Photothermal nanomaterial; RNA imaging; Rolling circle amplification.