Surface biopassivation of replicated poly(dimethylsiloxane) microfluidic channels and application to heterogeneous immunoreaction with on-chip fluorescence detection.
AuthorsLinder V, Verpoorte E, Thormann W, de Rooij NF, Sigrist H
JournalAnal Chem
PubMed ID11569807
Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization ... More
Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS).
AuthorsChen G, Hayhurst A, Thomas JG, Harvey BR, Iverson BL, Georgiou G
JournalNat Biotechnol
PubMed ID11385457
Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as ... More
Flow cytometric screening of cell-based libraries.
AuthorsDaugherty PS, Iverson BL, Georgiou G
JournalJ Immunol Methods
PubMed ID10986416
Flow cytometry is a powerful, high-throughput library screening tool in numerous applications including the isolation of bioactive molecules from synthetic combinatorial libraries, the identification of virulence genes in microorganisms, and the study and engineering of protein functions. Using flow cytometry, large libraries of protein mutants expressed in microorganisms can be ... More
Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.
AuthorsDaugherty PS, Olsen MJ, Iverson BL, Georgiou G
JournalProtein Eng
PubMed ID10436088
Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia ... More
Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.
AuthorsDaugherty PS, Chen G, Iverson BL, Georgiou G
JournalProc Natl Acad Sci U S A
PubMed ID10688877
Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) ... More