A signal-amplifiable biochip quantifies extracellular vesicle-associated RNAs for early cancer detection

Jiaming Hu,Yan Sheng,L James Lee,Junfeng Shi,Kwang Joo Kwak,Bohao Yu

doi:10.1038/s41467-017-01942-1

Abstract

Detection of extracellular vesicle (EV)-associated RNAs with low expression levels in early-stage cancer remains a challenge and is highly valuable. Here, we report a nanoparticle-based biochip that could capture circulating EVs without isolation, brighten encapsulated RNAs, and amplify fluorescence signals in situ in a single step. We confine catalyzed hairpin DNA circuit (CHDC) in cationic lipid-polymer hybrid nanoparticles (LPHNs) that are tethered on a chip. LPHN features a core-shell-corona structure that facilitates the transfer and mixing of CHDC with EV-associated RNAs when forming the LPHN–EV nanocomplex. CHDC is triggered upon target RNA binding and quickly generate amplified signals. The signal amplification efficiency of LPHN–CHDC is demonstrated in artificial EVs, cancer cells, and cancer cell-derived EVs. We show that LPHN–CHDC biochip with signal amplification capability could selectively and sensitively identify low expression glypican-1 mRNA in serum EVs, distinguishing patients with early- and late-stage pancreatic cancer from healthy donors and patients with benign pancreatic disease.

Highlights

Detection of extracellular vesicle (EV)-associated RNAs with low expression levels in earlystage cancer remains a challenge and is highly valuable
The lipid-polymer hybrid nanoparticles (LPHNs)–EV fusion leads to mixing of H1, H2, and reporter in the LPHN with RNAs in the EV
Using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), AsPC-1 cells with KRASG12D mutation were identified, while HPDE6-C7 control cells did not exhibit KRASG12D mutation (Fig. 3e). Both total internal reflection fluorescence (TIRF) images and fluorescence microscopy images revealed an intense fluorescence signal of KRASG12D expression in AsPC-1 cells, in contrast to a negligible signal in HPDE6-C7 cells, indicating the excellent selectivity of LPHN–catalyzed hairpin DNA circuit (CHDC) for KRASG12D mutation detection in cancer cells (Fig. 3f, d; Supplementary Fig. 10). These results demonstrate that LPHN–CHDC could achieve excellent image amplification of specific messenger RNA (mRNA) targets in living cells, allowing distinction of pancreatic cancer cells from normal pancreatic cells

Summary

Introduction

Detection of extracellular vesicle (EV)-associated RNAs with low expression levels in earlystage cancer remains a challenge and is highly valuable. EVs are membrane-enclosed vesicles of endocytic origin and contain proteins and nucleic acids[6,7,8,9,10]. In early-stage cancer, efficient quantification of EV-associated RNAs with low expression levels remains a challenge. It is vital to develop facile and inexpensive techniques that can capture individual EV and identify EV-associated RNA targets with high sensitivity and specificity for early cancer diagnosis. CHDC can generate multiple signal outputs when hybridized with target RNA to achieve signal amplification for effective quantification of RNAs with low copy numbers. We present a novel system termed signal-amplifiable LPHN–CHDC biochip capable of highly selective and sensitive quantification of target RNAs in EVs to achieve non-invasive early cancer diagnosis. Our findings indicate that the LPHN–CHDC biochip is a resourceful and simple to implement signal amplification tool for early cancer detection

Methods

Results

Conclusion