Development of a fluorescent quantum dot-mycolic acid probe for use in Mycobacterium tuberculosis antibody detection

Abstract

Tuberculosis (TB) is a highly contagious chronic pulmonary disease caused by Mycobacterium tuberculosis (M.tb). Early detection of this deadly disease using a simple and effective method at the point of care is not yet available, and this poses a serious challenge, especially in developing countries, because the start of treatment could be delayed allowing the disease to spread. Therefore, rapid, inexpensive, and accurate methods for early detection and diagnosis of TB are required. In this work, therefore, quantum dots (QDs) were coupled to mycolic acids (MAs) to investigate their potential to serve as water-soluble fluorescent tuberculosis (TB) biosensors (probes). MAs are found in the cell wall of Mycobacterium tuberculosis. They are lipids that are antigenic, interacting well with anti-MA antibodies, and are soluble only in chloroform and hexane. QDs are nanomaterials with attractive optical properties such as high fluorescence, good biocompatibility, and good water solubility. Water-soluble core/shell cadmium selenide/zinc sulphide quantum dots capped with L-cysteine (L-cys-CdSe/ZnS QDs) and amine functionalised graphene quantum dots (GQDs) were synthesized and covalently coupled (linked) to chloroform-soluble MAs via amide linkages to form water-soluble fluorescent probes: mycolic acid-cadmium selenide/zinc sulphide quantum dots (MA-CdSe/ZnS QDs) and mycolic acid-graphene quantum dots (MA-GQDs), respectively. The successful synthesis of these water-soluble fluorescent probes was confirmed by a series of spectroscopic methods including: electronic absorption spectroscopy (UV-Vis), fluorescence spectrophotometry, transmission electron microscopy (TEM), powder X-ray diffraction (XRD) spectroscopy, and Fourier-transform infrared (FT-IR) spectroscopy. Visual lateral flow of the coupled fluorescent probes was achieved on strips of nitrocellulose membrane using both water and membrane blocking solution as eluents, showing potential for development of a lateral flow device. To explore the possibility of interaction of the MA-QDs with TB biomarkers, the interaction between water-soluble fluorescent probes and the known synthetic anti-MA antibodies (gallibodies) was monitored by the enzyme-linked immunosorbent assay (ELISA) test, paper-based lateral flow assay, and fluorescence techniques. The coupled fluorescent probes (biosensors) showed good solubility in water, have high fluorescence, and visually flowed through a nitrocellulose membrane. However, preliminary tests indicated that the interaction between these water-soluble fluorescent probes and the anti-MA antibodies were non-specific. This work demonstrated that the main objective of improving the solubility of the coupled fluorescent probes was achieved, including that of coupled GQDs which are of lower toxicity than cadmium (Cd)-based QDs, although the specific binding with the anti-MA antibodies was not achieved. Future research recommendations to enhance the specific binding between the coupled fluorescent probes and the anti-MA antibodies include (i) increasing the amount of antigen MAs during the covalent coupling to QDs to improve the antigenic properties of the coupled materials, (ii) developing techniques that will be able to quantify the amount and concentration of MAs coupled to the QDs, and (iii) exploring the possibility of using molecular beacons (MBs) with M.tb DNA covalently attached to MA-QDs as a biosensor (probe) to increase the antigenic properties.