Sensitive and specific detection of PIK3CA point mutations using screen-printed electrochemical sensors with prior recombinase polymerase amplification

Beschreibung

Breast cancer is one of the most common causes of cancer-related death for women worldwide. A personalized treatment approach based on the non-invasive detection of point mutations in the PIK3CA gene enables efficient and precise anticancer therapy. Therefore, novel detection strategies are needed for rapid genetic diagnostics at the point of care (POC).
In the NanoPredict Project (grant number FO999899038), we target the sensitive and selective detection of the three predominant PIK3CA point mutations H1047R, E545K, and E542K, which are FDA-approved predictive biomarkers for the treatment of metastatic hormone receptor-positive, HER2-negative breast cancer patients with Alpelisib in combination with Fulvestrant. For this purpose, we developed a mutation-specific enzyme-linked electrochemical assay on multi-channel screen-printed gold sensors combined with isothermal recombinase polymerase amplification (RPA). For electrochemical detection, biotinylated deoxycytidine triphosphate (dCTP) was used to label the target DNA. This target DNA was generated employing either an asymmetric RPA approach or a symmetric RPA approach followed by lambda exonuclease digestion. The addition of a wild-type blocking DNA sequence during probe-target hybridization enables mutation-specific detection of PIK3CA point mutations.
As result, we could demonstrate the multiplex mutation-specific electrochemical detection of the amplified target sequences. The asymmetric RPA and the symmetric RPA with lambda exonuclease digestion led to comparable detection limits for the PIK3CA mutation H1047R with 229 copies/µL and 224 copies/µL, respectively. RPA with lambda exonuclease digestion, however, yielded higher currents and finally allowed for detecting the PIK3CA point mutations H1047R, E545K, and E542K in presence of wild-type target DNA at mutant allele fractions of >20%.
On the basis of these results, future activities will target the analysis of cell DNA of cancer tissues and cell-free DNA (cfDNA) in plasma samples with respect to the three PIK3CA mutations to study the feasibility of the electrochemical assay for liquid biopsy in clinical settings.

Zeitraum	13 Mai 2024
Ereignistitel	8th International Conference on bio-Sensing Technology
Veranstaltungstyp	Konferenz
Ort	Seville, SpanienAuf Karte anzeigen
Bekanntheitsgrad	International