Paper-based biomimetic test-strip for CA15-3 with coloured readout

Abstract

The combination of molecularly-imprinted polymers (MIPs) on paper substrates for capturing a cancer antigen 153 (CA15-3) with the traditional coloured transduction scheme of enzyme-linked immunosorbent assay (ELISA) assays using 3,3,5,5-tetramethylbenzidine (TMB), horseradish peroxidase (HRP) and H2O2 is presented here for the first time. Here, the paper surface was modified with a silane derivative containing an amine function that allows subsequent binding of 3-aminophenylboronic acid (3-APBA). The target protein, CA15-3, was then bound via the boronic groups of APBA. The empty space around CA15-3 was filled by polymerization of dopamine. Finally, the CA15-3 template was removed by breaking the imine function to create vacant sites for which CA15-3 has a high affinity. Binding of CA15-3 was detected by oxidation of TMB substrate by HRP in the presence of H2O2. The results showed that the MIP was able to selectively recognize CA15-3 within 3500 U mL-1, even in complex samples. Quantitative data were extracted by analysing the colour coordinates of images captured with a smartphone using ImageJ. This selective behaviour was confirmed by comparison with a control material (non polymer, NIP). Overall, the described approach has advantages over conventional ELISA, namely low cost and high stability, which is due to the use of MIP as a trapping element acting as a selective pre-concentration point of CA15-3. To our knowledge, this is the first biomimetic ELISA (B-ELISA) on paper substrate used for macromolecules such as proteins. We believe that this approach has the potential to be applied to other protein disease biomarkers, making it a suitable tool for screening glycoproteins at the point-of-care.