Distinguishing PIK3CA p.E545K Mutational Status from Pseudogene DNA with a Next-Generation ISFET Sensor Array

PIK3CA p.E545K mutation is a well-studied breast cancer biomarker with a clinical significance as a therapeutic target, particularly with the use of the small molecule inhibitor Alpelisib. An issue with detecting this mutation and other mutations in this exon is that 98% of its sequence homology is identical to a pseudogene, a non-functional non-coding gene found in chromosome 22. This paper aims to use ISFET enabled Lab-on-Chip (LoC) technology, coupled with a well-studied isothermal amplification method (LAMP), as a means to distinguish wild-type (WT), mutant (MT) and pseudogene (PG) copies of DNA. In an age where affordability and accessibility of diagnostic tests is of crucial importance, the use of CMOS technology offers a great potential as an alternative for regular near-patient molecular testing using liquid biopsies. Bespoke primer design is tested and optimised with synthetic DNA to achieve high specificity and low sensitivity (100 copies). The primer efficiencies were also tested on our in-house LoC system showing near identical results to those obtained from a qPCR instrument. Spiking experiments were also conducted, where mixed populations of WT and MT were tested to assess the primers' abilities to estimate the variant allele frequency (VAF) of p.E545K and to mimic clinical scenarios. The results continue to depict how LoC technology in partnership with LAMP detection can be used in a liquid biopsy setting to detect blood DNA biomarkers to assist better patient stratification.