Rapid, sensitive, and label-free impedimetric detection of a single-nucleotide polymorphism correlated to kidney disease

We present a protocol for the very rapid and sensitive detection of a specific mutation of the COL4A5 gene (exon 29, A-C mismatch) which was found in people affected by Alport syndrome (AS) and their families. Disposable electrochemically printed electrodes were used to immobilize a single-stranded oligonucleotide probe that was complementary to the AS-correlated gene. The detection principle is based on changes in the impedance spectra of the redox probe ferro/ferricyanide after hybridization with synthetic target DNA. Detection was performed either for mutated or for healthy (wild-type) gene copies. The high sensitivity obtained with this protocol (LOD in the picomolar range) was additionally enhanced to the femtomolar range by performing the detection in the presence of Ca²⁺. In fact, the specific binding of the metal ions in the presence of an A-C nucleotide mismatch induced a further impedance change, thus improving the discrimination between the mutated and healthy gene, as the signal amplification is achieved only for the former.