An electrochemical molecular assay for the detection of cancer-associated Thomsen-Friedenreich glycans.

person Saimon Moraes Silva Swinburne University of Technology, Melbourne, Australia info_outline Saimon Moraes Silva, Chathurika Abeyrathne, Miaosi Li, Luke Robert Cossins, Anushka Samudra, Wren Greene, Simon Moulton

Authors person Saimon Moraes Silva Swinburne University of Technology, Melbourne, Australia info_outline Saimon Moraes Silva, Chathurika Abeyrathne, Miaosi Li, Luke Robert Cossins, Anushka Samudra, Wren Greene, Simon Moulton Organizations Swinburne University of Technology, Melbourne, Australia, Universal Biosensors, Melbourne, VIC, Australia, Universal Biosensors, Rowville, Australia, Deakin University, Melbourne, VIC, Australia, Swinburne University, Melbourne, VIC, Australia Abstract Disclosures Research Funding Other Government Agency Australian Research Council, Universal Biosensors Background: Abnormal glycosylation is a fundamental characteristic in cancer cells, where expression of glycans such as Thomsen-Friedenreich (T), Tn, and sialyl-Tn (STn) are related to particular tumour patterns. T, Tn and STn antigens are unusually short and truncated O-glycans expressed on glycoproteins synthesized within cancer cells and not normally found in healthy tissue or circulating within the blood. The association between blood circulating T-glycans and cancer progression and poor prognosis is now well-known and highlights great potential as biomarkers for cancer screening, diagnosis, and surveillance of diseases during treatment and following remission. Despite this potential, there is currently no established molecular assay for detecting or quantifying these biomarkers in blood biopsy samples. Methods: Using a test strip format reminiscent of a blood glucose test, a new electrochemical molecular assay is introduced for the detection of short O-glycans directly in unprocessed bodily fluids. This assay is based on a self-assembling sensing interface consisting of two glycoproteins, recombinant human lubricin (LUB) and peanut agglutin (PNA). In this interface, the LUB functions as both an anti-fouling layer which mitigates electrode passivation in blood and as a crucial component in the biosensing mechanism for generating readable electrical signals in the presence of target glycans. The PNA, a plant-based lectin, presents high binding specificity towards the T glycoform and functions as the sensor recognition element which is bound to the LUB through T glycans naturally present in the highly glycosylated protein backbone. In this sensing interface, electrical signals are generated by engineering a redox reporter, methylene blue into the LUB/PNA interfacial complex, where the electrical signal is altered by the capture of target glycans by PNA and its impact on the molecular dynamics of the LUB/PNA interfacial complex. Results: Using this test strip, it is possible to detect short cancer-related glycans in concentrations as low as nanomolar directly in small volumes of unprocessed whole blood. These test strips likewise show comparable results to an ELISA test developed in parallel for the detection of short O-glycans. The tests carried out with retrospective blood plasma samples from patients presenting three different tumour types, including prostate (18 patients), colorectal (15 patients), and breast cancer (6 patients), and plasma samples from healthy donors (10 different donors) showed differentiation of response signals between healthy and diseased patients. Conclusions: These findings highlight the clinical potential of this new disposable test strip for the detection of tumour associated glycans, which can be incorporated into point-of-care devices, for cancer monitoring and perhaps one day in screening and diagnosis.