Synergy of Peptide–Nucleic Acid and Spherical Nucleic Acid Enabled Quantitative and Specific Detection of Tumor Exosomal MicroRNA

Abstract

Exosomal microRNAs are essential in intercellular communications and disease progression, yet it remains challenging to quantify the expression level due to their small size and low abundance in blood. Here, we report a “sandwich” electrochemical exosomal microRNA sensor (SEEmiR) to detect target microRNA with high sensitivity and specificity. In SEEmiR, neutrally charged peptide nucleic acid (PNA) enables kinetically favorable hybridization with the microRNA target relative to negatively charged DNA, particularly in a short sequence (10 nt). More importantly, this property allows PNA to cooperate with a spherical nucleic acid (SNA) nanoprobe that heavily loads with oligonucleotide-adsorbed electroactive tags to enhance detection sensitivity and specificity. Such a PNA–microRNA–SNA sandwich construct is able to minimize the background noise via PNA, thereby maximizing the SNA-mediated signal amplification in electrostatic adsorption-based SEEmiR. The synergy between PNA and SNA makes the SEEmiR sensor able to achieve a broad dynamic range (from 100 aM to 1 nM) with a detection limit down to 49 aM (2 orders of magnitude lower than that without SNA) and capable of distinguishing a single-base mismatch. This ultrasensitive sensor provides label-free and enzyme-independent microRNA detection in cell lysates, unpurified tumor exosomal lysates, cancer patients’ blood, and accurately differentiates the patients with breast cancer from the healthy ones, suggesting its potential as a promising tool in cancer diagnostics.

Exosomes as a Source of Cancer Biomarkers: Advances in Electrochemical Biosensing of Exosomes

“…In addition to CD63, quite a number of other exosomal proteins were targeted for biosensing purposes involving exosome‐surface‐confined proteins like EpCAM, CD24, CA125, HER2, MUC18, PSMA, FAM134B, GPC1, AFP, CEA, CD9 or exosomal phosphatidylserine (Table ). A typical cancer biomarker detected in the intra‐exosomal space (exosome interior i. e. exosome cargo) was miRNA‐21 and only one protein to date . In order to detect biomarkers from the intra‐exosomal space, detergents are usually used, but there is a possibility to induce lysis of vesicles using an electric field, making it possible to integrate lysis and the analysis of biomarkers present in the intra‐exosomal space even without surfactants.…”

“…Furthermore, CRC‐derived cell lines contain several proteins on the surface of exosomes, which can be applied as cancer biomarkers such as cytokeratin 19, tumour‐associated glycoprotein 72 and carbohydrate antigen 125 . Label‐free detection of miRNA‐21 was also performed using PNA (peptide nucleic acid) immobilised on a gold electrode . miRNA‐21 (22 nucleotides) released from exosomes produced by two cell lines was hybridised with immobilised PNA (10 nucleotides, no negative charge) and in the final step a sandwich configuration was formed by incubation of the modified electrode with spherical nucleic acid (SNA).…”

“…The sensor could detect miRNA‐21 down to 49 aM and without SNA a much higher LOD of 9.2 fM was observed. The biosensor was used in the analysis of exosomal miRNA‐21 from 6 healthy individuals and 6 breast cancer patients with a good discrimination power …”

“…A large number of Ru(NH 3 ) 6 3+ cations bind electrostatically to negatively charged phosphate moieties of densely assembled DNA strands in a stoichiometric fashion, which makes it possible to quantitatively reflect the amount of DNA confined on the electrode by chronocoulometric interrogation of the redox reaction of Ru(NH 3 ) 6 3+ . Reprinted with permission from . Copyright (2019) American Chemical Society.…”

Chemical binding of pyrrolidinyl peptide nucleic acid (acpcPNA‐T9) probe with AuNPs toward label‐free monitoring of miRNA‐21: A novel biosensing platform for biomedical analysis and POC diagnostics

“…16 It presents unique advantages such as stability against chemicals, proteases, and nucleases compared with natural oligonucleotides, hence enhances the efficiency of hybridization. 17 Pyrrolidinyl PNA with D-prolyl-2-aminocyclopentanecarboxylic backbone (acpcPNA) exists unique properties for binding to RNA such as qualified biological stability and a highly sequence-specific manner with superior mismatch discrimination capacities than other DNA probes. These advantages of acpcPNA provide a promising platform for genosensors.…”