Immobilization-free multiplex electrochemical DNA and SNP detection

A novel electrochemical method for multiplex detection of sequence-specific DNA and single nucleotide polymorphism (SNP) that requires no probe immobilization is reported. The immobilization-free detection of target DNA is realized by the use of a neutrally charged peptide nucleic acid (PNA) probe labeled with an electroactive indicator and a negatively charged ITO electrode. Upon hybridization between the target DNA and the complementary PNA probe, electrostatic repulsion between the negative backbone of the DNA/PNA duplex and the negative surface of the ITO electrode prevents the electroactive indicator from approaching the electrode, resulting in a significantly suppressed electrochemical signal. Multiplex DNA or SNP detection is enabled by using multiple PNA probes with different sequences labeled with different distinguishable electroactive indicators. Due to the immobilization-free nature of this detection scheme, no interference is found between the simultaneous detection of multiple target DNAs or SNPs. This simple and robust immobilization-free multiplex DNA and SNP detection strategy may find applications in a wide range of fields especially in point-of-care testing.