Multiplex electrochemical sensing platforms for the detection of breast cancer biomarkers

Herein, advancements in electroanalytical devices for the simultaneous detection of diverse breast cancer (BC) markers are demonstrated. This article identifies several important areas of exploration for electrochemical diagnostics and highlights important factors that are pivotal for the successful deployment of novel bioanalytical devices. We have highlighted that the limits of detection (LOD) reported for the multiplex electrochemical biosensor can surpass the sensitivity displayed by current clinical standards such as ELISA, FISH, and PCR. HER-2; a breast cancer marker characterised by increased metastatic potential, more aggressive development, and poor clinical outcomes; can be sensed with a LOD of 0.5 ng/ml using electrochemical multiplex platforms, which falls within the range of that measured by ELISA (from picogram/ml to nanogram/ml). Electrochemical multiplex biosensors are reported with detection limits of 0.53 ng/ml and 0.21 U/ml for MUC-1 and CA 15-3, respectively, or 5.8 × 10^-3 U/ml for CA 15-3 alone. The sensitivity of electrochemical assays is improved when compared to conventional analysis of MUC-1 protein which is detected at 11-12 ng/ml, and ≤30 U/ml for CA 15-3 in the current clinical blood tests. The LOD for micro-ribonucleic acid (miRNA) biomarkers analyzed by electrochemical multiplex assays were all notedly superior at 9.79 × 10^-16 M, 3.58 × 10^-15 M, and 2.54 × 10^-16 M for miRNA-155, miRNA-21, and miRNA-16, respectively. The dogma in miRNA testing is the qRT-PCR method, which reports ranges in the ng/ml level for the same miRNAs. Breast cancer exosomes, which are being explored as a new frontier of biosensing, have been detected electrochemically with an LOD of 10³-10⁸ particles/mL and can exceed detection limits seen by the tracking and analysis of nanoparticles (∼ 10⁷ particles/ml), flow cytometry, Western blotting and ELISA, etc. A range of concentration at 78-5,000 pg/ml for RANKL and 16-1,000 pg/ml for TNF is reported for ELISA assay while LOD values of 2.6 and 3.0 pg/ml for RANKL and TNF, respectively, are demonstrated by the electrochemical dual immunoassay platform. Finally, EGFR and VEGF markers can be quantified at much lower concentrations (0.01 and 0.005 pg/ml for EGFR and VEGF, respectively) as compared to their ELISA assays (EGRF at 0.31-20 ng/ml and VEGF at 31.3-2,000 pg/ml). In this study we hope to answer several questions: (1) Are the limits of detection (LODs) reported for multiplex electrochemical biosensors of clinical relevance and how do they compare to well-established methods like ELISA, FISH, or PCR? (2) Can a single sensor electrode be used for the detection of multiple markers from one blood drop? (3) What mechanism of electrochemical biosensing is the most promising, and what technological advancements are needed to utilize these devices for multiplex POC detection? (4) Can nanotechnology advance the sensitive and selective diagnostics of multiple BC biomarkers? (5) Are there preferred receptors (antibody, nucleic acid or their combinations) and preferred biosensor designs (complementary methods, sandwich-type protocols, antibody/aptamer concept, label-free protocol)? (6) Why are we still without FDA-approved electrochemical multiplex devices for BC screening?