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

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.