Development of a Multifunctional Nanobiointerface Based on Self-Assembled Fusion-Protein rSbpA/ZZ for Blood Cell Enrichment and Phenotyping.

Abstract

We present a multifunctional nanobiointerface for blood cell capture and phenotyping applications that features both excellent antifouling properties and high antibody activity. Multifunctionality is accomplished by modifying polymeric materials using self-assembled S-layer fusion-protein rSbpA/ZZ to immobilize high density antibodies at the two protein A binding sites of the rSbpA/ZZ nanolattice structure. Controlled orientation and alignment of the antibodies reduced antibody consumption 100-fold and increased cell capture efficiency 4-fold over standard methodologies. Cell analysis in complex samples was made possible by the remarkable antifouling properties of the rSbpA domain, while at the same time reducing unspecific binding and forgoing tedious blocking procedures. An automated microfluidic in situ cell analysis platform for isolation and phenotyping of primary peripheral blood mononuclear cells was developed as practical application. Results obtained using our automated microfluidic cell analysis platform showed that the multifunctional nanobiointerface can discriminate among T helper and cytotoxic T cells, and thymocytes. Additionally, on-chip cell capture under flow conditions using a high affinity CD 3 selective nanobiointerface preferentially isolated cells with strong surface marker expression. This means that our dynamic microfluidic cell purification method allows the enrichment of 773 CD 8 positive cytotoxic T cells out of a total blood cell population of 7728 PBMCs, which is an increase in cell enrichment of 8-fold with a purity of 85%.