A sensitive paper-based lateral flow immunoassay platform using engineered cellulose-binding protein linker fused with antibody-binding domains

Abstract

An advanced paper-based LFIA using engineered cellulose-binding protein linker fused with antibody-binding domains was developed to resolve problems associated with conventional LFIA strip, including the limitation of quantitative analysis and low sensitivity. CBP31-BC linker was prepared by genetically fusing the cellulose binding modules of family 3 (CBM3) and family 1 (CBM1) with antibody-binding B and C domains of Protein A. Cellulose-binding analysis showed that the addition of two CBMs led to high cellulose-binding capacity in CBP31-BC. Transmission electron microscopy and quartz crystal microbalance analyses demonstrated that the cellulose-binding ability of CBP31-BC enabled the BC domain to be efficiently exposed, allowing about 6-fold higher antibody-binding efficiency (similar to 32.5 %) than that (similar to 5.5 %) of sole BC. To evaluate the feasibility of the CBP31-BC linker-employed LFIA platform, an immunoassay of prostate-specific antigen (PSA) was performed as a model reaction. The CBP31-BC-employed paper-based LFIA detected PSA at levels as low as 0.25 ng/mL in 20 min, which is about 10-fold more sensitive to conventional LFIAs that use simple physical adsorption. CBP31-BC-employed LFIA also exhibited a lower detection range of 0.25-2.5 ng/mL when compared to conventional LFIAs. These results might be due to efficient oriented immobilization of the capture antibody by CBP31-BC linker on cellulose membrane. Thus, our developed CBP31-BC linker-employed paper-based LFIA platform holds great promise for the detection of target markers due to significantly enhanced sensitivity.