Ultrasensitive Biosensors for Quantification of Multiple Immune Checkpoint Inhibitor Biomarkers from Blood, Plasma and Serum with Investigation of Protein Corona Formation and its Implications

Abstract

The expression levels of immune checkpoint inhibitor biomarkers including programmed death-ligand 1 (PD‐L1), cytokine lymphocyte antigen 4 (CTLA-4) and B7-homolog3 (B7-H3) can be used to identify the presence of diseases, and also show a good correlation with therapeutic outcomes. Invasive solid biopsies are used to achieve samples with immunohistochemistry assays being the detection method of choice to identify the expression levels of these biomarkers in clinical practice. These assays are qualitative or semi-quantitative due to biopsy heterogeneity. There is an unmet clinical need for quantitative detection methods that are less invasive, to improve the efficacy of treatments that are closely associated with PD-L1, CTLA-4 and B7-H3 expression levels. The detection of PD-L1, CTLA-4 and B7-H3 in whole blood is an attractive pathway for the early detection, prediction and evaluation of cancer treatment response because of its simplicity but remains almost completely unexplored. The challenge is that a limit of detection of less than picomolar must be achieved for a detection technology to satisfy the requirements of the unmet need. The purpose of this work is firstly to address the unmet need for improved conventional immune checkpoint inhibitor biomarkers detection techniques by designing an ultrasensitive quantitative biosensor based on gold-coated magnetic nanoparticles, referred to as dispersible electrodes. The research demonstrates the ultrasensitive, selective and rapid electrochemical detection of PD-L1, CTLA-4 and B7-H3 directly in whole blood. These dispersible electrodes selectively capture analytes within biofluids and upon application of a magnet, they are reassembled into a macroelectrode for electrochemical detection of the target antigen using a classic sandwich immunoassay with a detection range of nanomolar to attomolar and a response time of only 15 min. The research then focuses on how this system is capable of detecting these species in whole blood without being completely fouled by proteins. Investigations show that ‘soft protein corona’ layer forms around the antibody-modified particles, which can be resulted in lower signal intensity and greater uncertainties of dispersible electrodes but does not completely suppress electrochemistry.