Combinatorial Probes for High-Throughput Electrochemical Analysis of Circulating Nucleic Acids in Patient Samples

The analysis of circulating tumour nucleic acids (ctNAs) provides a minimally invasive way to assess the mutational spectrum of a tumour. However, effective and practical methods for analyzing this emerging class of markers are lacking. Analysis of ctNAs using a sensor-based approach has notable challenges, as it is vital to differentiate nucleic acids from normal cells from mutation-bearing sequences emerging from tumours. Moreover, many genes related to cancer have dozens of different mutations. Here, we report an electrochemical approach that directly detects genes with mutations in patient serum by using combinatorial probes (CPs). The CPs enable detection of all of the mutant alleles derived from same part of the gene. As of proof-of-concept we analyze mutations of the EGFR gene, which has more than 40 clinically-relevant alterations that include deletions, insertions, and point mutations. Our CP-based approach accurately detects mutant sequences directly in patient serum. Noninvasive analysis of circulating tumour-derived nucleic acids (ctNAs) is an appealing approach for cancer monitoring, as serial blood draws are possible for repetitive and longitudinal sampling, while solid tumours require invasive biopsies.1 However, reliable detection of nucleic acids containing mutations that allow specific detection of ctNAs is very challenging as patient samples contain a very small percentage of mutated nucleic acids in a large background of normal nucleic acids. Analysis of mutated nucleic acids in the blood, for example the EGFR (epidermal growth factor receptor) and KRAS (kirsten rat sarcoma viral oncogene homolog) genes, could allow specific monitoring of cancer-related sequences.2 However, for detection of these mutated ctNAs, a very sensitive and specific method is required, as mutated ctNAs are present along with a high level of normal sequences. Currently, the most commonly used methods for ctNA analysis are DNA sequencing3 and the polymerase chain reaction (PCR).4 DNA sequencing is a powerful technique for research studies, but its application is restricted due to the high cost for routine clinical use, and long turnaround time (2-3 weeks).3d Although conventional PCR methods can’t detect mutated ctNAs as they cannot detect minor variants at levels < 20%, some PCR-based methods, such as allele-specific clamp PCR, COLD-PCR (co-amplification at lower denaturation temperature-PCR), and digital PCR have successfully detected ctNAs.4 However, PCR methods are susceptible to false negatives and positives produced by interference from chemical species present in clinical samples; the use of this approach therefore requires trained personnel and preprocessing of samples including purification of nucleic acids. This requirement limits the use of this technique to clinical laboratories. Thus, a PCR-free method that is able to detect mutated ctNAs directly in serum or blood is urgently needed to allow liquid biopsies monitoring ctNAs to become more routine. Chip-based electronic and electrochemical methods have been pursued as a promising alternative for clinical sample analysis because they can be automated and do not rely on costly instrumentation.5 Particularly, electrochemical methods have received attention because of their low cost, high sensitivity, and amenability to high levels of multiplexing.6 Electrochemical techniques have been employed successfully to analyze various tumour markers,7 and infectious pathogens,8 but the analysis of ctNAs for tumour-related mutations in patient samples is a new application for this type of analysis that was first described less than three years ago.9 An electrochemical strategy developed by our laboratory for the analysis of a small set of point mutations in ctNAs was one of the first to address how a chip-based approach could facilitate ctNA analysis.9 High-surface area, three-dimensional microelectrodes were functionalized with probe sequences complementary to a sequence of interest, and hybridization of targets was read out with an electrocatalytic reporter strategy. PNA clamp molecules were used to limit cross-reactivity with wildtype nucleic acids and other mutated sequences. The technique specifically detected mutated ctNAs at physiologically-relevant levels in 30 40 minutes. However, the KRAS and BRAF genes only contain point mutations in ctNAs. There many other sequence alterations that may appear in ctNAs. For example, EGFR, a sequence that is often mutated in lung cancer tumours, not only contains point mutations but also deletions and insertions.10,11 However, analysis of EGFR is very challenging as it has more than 40 clinically-relevant sequence changes.12 [a] Dr. J. Das, Dr. I. Ivanov, Prof. S. O. Kelley Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy University of Toronto Toronto, ON M5S 3M2, Canada E-mail: shana.kelley@utoronto.ca [b] Dr. T. S. Safaei, Prof. E. H. Sargent Department of Electrical and Computer Engineering, Faculty of Engineering Department University of Toronto Toronto, ON, Canada