A rapid bioanalytical tool for detection of sequence-specific circular DNA and mitochondrial DNA point mutations.

Mutations in mitochondrial DNA (mtDNA) have been an essential cause of numerous diseases, making their identification critically important. The majority of mtDNA screening techniques require polymerase chain reaction (PCR) amplification, enzymatic digestion, and denaturation procedures, which are laborious and costly. Herein, we developed a sensitive PCR-free electrokinetic-based sensor combined with a customized bis-peptide nucleic acid (bis-PNA) and gamma-PNA (γ-PNA) probes immobilized on beads, for the detection of mtDNA point mutations and sequence-specific supercoiled plasmid DNA at the picomolar range. The probes are capable of invading the double-stranded circular DNA and forming a stable triplex structure. Thus, this method can significantly reduce the sample preparation and omit the PCR amplification steps prior to sensing. Further, this bioanalytical tool can open up a new paradigm in clinical settings for the screening of double-stranded circular nucleic acids with a single-base mismatch specificity in a rapid and sensitive manner.

Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients.

Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALK-tyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.