Non-enzymatic detection of miR-21 in cancer cells using a homogeneous mix-and-read smart probe assay

We report a new assay system for the detection of miR-21 in cancer cells. The new assay works at room temperature and it does not involve enzymatic amplification. It consists a hairpin smart probe, designed to specifically recognize miR-21 target sequence. We tested the performance and sequence recognition capability of the smart probe to confirm desired specifications. We used the smart probe for the sequence-specific recognition of synthetic miR-21 oligonucleotides as well as mismatch sequences and we found that the probe recognizes the target sequence-specifically, while discriminating against mismatched sequences. We determined the limit of detection and limit of quantitation for the miR-21 oligonucleotides to be 1.72 nM and 5.78 nM, respectively, while the sensitivity is 6.90 × 10¹¹ c.p.sM⁻¹. More importantly, we showed that the smart probe-based method is also sensitive and selective for miR-21 when applied to crude extractions from MCF-7 cancer cell line at room temperature, with the results showing high fluorescence signals for the MCF-7 samples while showing much less signals for samples that did not contain miR-21. Thus, this new smart probe system constitutes a homogeneous, mix-and-read detection technique that can provide reliable diagnostics of miR-21 cancer biomarker at room temperature.