Towards the development of Molecularly Imprinted Polymer (MIP) for lineage specific cell surface antigens used in cancer diagnosis.

Abstract

Immunohistochemistry using antibodies plays a pivotal role in the diagnosis of various solid cancers and haematological malignancies such as leukaemias, lymphomas and myelomas. However, antibodies have a number of disadvantages including its high cost, requirement for refrigeration for transport and storage and its limited shelf life. Hence, the search for a sensitive and specific diagnostic platform that is robust and reproducible and one that utilises a capture ligand which is easy and cheap to manufacture and is stable at room temperature with a long shelf life. Such diagnostic platform would be particularly useful in developing countries, where facilities for storage and transportation at sub-zero temperatures are limited. Molecularly Imprinted Polymers (MIPs), one of the rapidly advancing technologies for nanodiagnostics, may offer such a solution in cancer diagnostics. This exploratory study was undertaken to investigate whether MIP nanoparticles synthesised using the solid-phase approach and epitope imprinting method have potential to replace antibodies in cancer diagnosis. The common leucocyte antigen or CD45 protein which is universally expressed on haemopoietic cells was chosen as the candidate for molecular imprinting because the expression of this antigen can differentiate blood cancers from other neoplasia. In order to make the process cost-effective, a custom-made peptide template with the amino acid sequence that is widely used for anti-CD45 antibody production was used for imprinting. A modification to the amino acid sequence of the template was made by adding the amino acid cysteine which has a thiol group, to the carboxyl end of the CD45 template peptide and anchored to silica nanoparticles to improve the homogeneity of the imprinted polymers. Synthesis of MIPs was carried out using two different compositions of functional monomers, one with a 'standard' mix of monomers and the other one containing a fluorinated monomer. The characterisation of the synthesised nanoMIPs and the binding of the target protein to the MIPs were studied using dynamic light scattering (DLS) and tunable resistive pulse sensing (TRPS). The results of this study prove that the solid-phase synthesis using a custom made polypeptide as the template (segment imprinting) is a logical approach. One important technical refinement is the immobilisation of the template protein to the silica beads in a single orientation via the amino acid cysteine. This modification resulted in the production of more uniform nanoMIPs with low polydispersity. Another significant observation of this study is that the use of fluorinated monomer in combination with the 'standard functional monomers' for the MIP synthesis has improved the quality of the nanoMIPs produced. Furthermore, this study has successfully explored, for the first time, the usefulness and applicability of the technique of tunable resistive pulse sensing (TRPS) for the characterisation of nanoMIPs. The preliminary results obtained in this study indicates that this technique may be superior to dynamic light scattering (DLS) for not only measuring the size and size distribution of the particles but also to study MIP-target interactions. The TRPS analysis of the changes in the zeta potential of the nanoMIPs has shown that the CD45 epitope imprinted nanoMIPs bind to the CD45 protein.