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

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.Life and Consumer SciencesPh. D. (Life and Consumer Sciences

Synthesis, density functional theory, molecular dynamics and electrochemical studies of 3-thiopheneacetic acid-capped gold nanoparticles

Gold nanoparticles capped with a bifunctional ligand, 3-thiopheneacetic acid (3-TAA) were synthesised by borohydride reduction at room temperature. The transmission electron microscopy (TEM) analysis showed that the particle aggregates and had semi-linear partial linkages that could be attributed to multi-modal binding of the ligand with various gold nanoparticles through the terminal thiolether (–S–) group and oxygen of the carboxylic (–COOH) group. This bimodal interaction led to limited stability of the resultant nanoparticles when tested using highly electrolytic media. To investigate further, density functional theory (DFT) quantum chemical and molecular dynamic calculations were conducted. The energetically favorable binding modes of the ligand to gold nanoparticle surfaces using the Gaussian program were studied. The DFT results showed kinetic stability of Au–3-TAA–Au interactions leading to inter-particle coupling or aggregation. Electrochemical analysis of the resultant nature of the capping agent revealed that 3-thiopheneacetic acid did not form a polymer during the preparation of Au–3-TAA. The cyclic voltammograms of Au–3-TAA nanoparticles coated glassy carbon electrode showed a typical gold character with the oxidation and reduction peaks at 1.4 V and 0.9 V, respectively

Synthesis of gold and palladium thiolato complexes and their applications as sulfur dioxide sensors

M.Sc.[AuCl(PPh3)] was reacted with mixed thiols in the presence of silver(I) oxide, resulting in complexes of the type [Au(SC6H4X)(PPh3)] X= Cl, NH2,CH2, forming silver chloride as a by-product. In addition to the above series [Au(SCH2(C6H4)3(2-C6H5(C6H4N)] was prepared via a different route, where [AuCl3(2-C6H5(C6H4N)] was reacted with benzyl mercaptan under reflux in the presence of silver(I) oxide for 3 h, forming silver chloride as a by-product. Palladium complex [PdCl2(2-C6H5(C6H4N)] was prepared by reacting [PdCl2(MeCN)] with 2-phenylpyridine at room temperature for 2 h. All complexes were characterized by 1H, 13C, 31P{H} NMR, IR, mass spectrometry and elemental analysis. Characterization of the starting materials [AuCl3(2-C6H5(C6H4N)] and [PdCl2(2- C6H5(6H4N)] by single crystal X-ray diffraction confirmed their chemical formula. All complexes were reacted with sulfur dioxide (SO2) and the reactions were monitored by electrochemistry and UV-vis spectroscopy. The electrochemical study of the complexes, using cyclic voltammetry (CV) and Osteryoung square wave voltammetry (OSWV), showed one anodic peak, which is due to gold(I/III) and an unresolved peak due to thiolate ligand. Upon bubbling of SO2 to the complexes, there was an immediate change of colour from clear to yellow, the CV results showing an increase in current of the gold(I/III) peak. UV-vis spectroscopy studies showed a shift of peak form 250-286 nm, upon bubbling of SO2 to complexes

A Review of the Nucleic Acid-Based Lateral Flow Assay for Detection of Breast Cancer from Circulating Biomarkers at a Point-of-Care in Low Income Countries

The current levels of breast cancer in African women have contributed to the high mortality rates among them. In South Africa, the incidence of breast cancer is also on the rise due to changes in behavioural and biological risk factors. Such low survival rates can be attributed to the late diagnosis of the disease due to a lack of access and the high costs of the 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 at an early stage. 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 of breast cancer can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC), especially in low-income countries (LICs). Nucleic-acid-based lateral flow assays (NABLFAs) that combine molecular detection with the immunochemical visualisation principles, have recently emerged as tools for disease diagnosis, even for low biomarker concentrations. Detection of circulating genetic biomarkers in non-invasively collected biological fluids with NABLFAs presents an appealing and suitable method for POC testing in resource-limited regions and/or LICs. Diagnosis of breast cancer at an early stage will improve the survival rates of the patients. This review covers the analysis of the current state of NABLFA technologies used in developing countries to reduce the scourge of breast cancer