Development of a nucleic acid-based lateral flow assay (NABLFA) for easy detection of breast cancer

Abstract

Women in Africa currently have the lowest registered cases of breast cancer. However, they have the highest mortality rates. In South Africa (SA), women are at a greater risk of been diagnosed with breast cancer due to changes in behavioural and genetical risk factors. Such high mortality rates can be attributed to the late diagnosis of the disease due to inaccessibilityand the high cost of current diagnostic tools. Breast cancer is asymptomatic, at early stages, which is the best time to detect it and intervene to prevent high mortality rates. Proper risk assessment, campaigns and access to adequate healthcare need to be prioritised among patients. Early detection of breast cancer can significantly improve the survival rate of breast cancer patients since therapeutic strategies are more effective at this stage. Early detection can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC) especially, in low-income countries (LIC). Nucleic acid-based lateral flow assays (NABLFA) methods that combine molecular detection with the immunochemical visualisation principles have recently emerged as tools to use for disease diagnosis even at low biomarker concentrations. Developing a NABLFA offers a rapid, simple, sensitive and inexpensive tool disease detection at POC. In this study, three known breast cancer target genes: tumour suppressor and transcription factor (p53), phosphatase and tensin homolog (PTEN) and breast cancer gene ( BRCA1) were identified through literature mining as molecular targets. The expression of these genes in various human cell lines was validated through bioinformatics and real-time polymerase chain reaction (R -PCR). DNA was extracted from MCF7, T4D7, MDA231, MCF12A,BHK-21, A375 and Me-180 cell lines using Wizard® Genomic DNA purification kit and amplified by conventional PCR using two sets gene-specific primers; with one set tagged with biotin (forward primer) and digoxigenin (dig, reverse). The NABLFA was developed by immobilising dig antibody (anti-dig) on the test line and biotin on the control line on a nitrocellulose membrane. The 14nm citrate-capped gold nanoparticles (AuNPs) were used as a colorimetric label. AuNPs were synthesised using citrate reduction method and characterised by UV-Vis absorption spectroscopy and transmission electron microscopy (TEM). Then conjugated with streptavidin using biotin-streptavidin chemistry and used as detection probe in the NABLFA. The binding and sensitivity of the analyte (biotin-tagged PCR product) to anti-dig was accessed by wet testing. The LFA strip was dipped in a tube containing biotin-tagged PCR product and AuNP-streptavidin conjugate. The binding of analyte to anti-dig was visually monitored after 15mins incubation with a show of red lines on both control and test lines. In silico analysis demonstrated that p53 and BRCA1 are highly expressed in breast cancer than normal tissues whereas PTEN is highly expressed in normal breast tissues than cancerous. This was further confirmed by conventional and real-time quantitative PCR (RT-PCR). The developed NABLFA successfully detected the PCR amplicons of p53 and PTEN validated by the appearance of two red lines both the control and the test line indicating the presence of the test biomarker. A rapid AuNPs-based NABLFA was successfully developed with a turn-around time of 10mins. The assay had a limit of detection of 0.06ng/ml and 0.125ng/ml for p53 and PTEN respectively. The AuNP-based NABLFA could serve as a screening tool for various diseases in resource-limited and LIC. Furthermore, in resource-limited areas, the assay can be developed into loop-mediated isothermal amplification (LAMP) diagnostic test to eliminate the use of conventional PCR in POC settings.