Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors

Abstract

Abstract:Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components ("BioBricks") provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell "renewable" capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and goldbinding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats.

Methodology for preservation of yeast-bound single chain fragment variable antibody affinity reagents

“…However, there are increasing numbers of in vitro approaches to generate similar binder molecules including small binder molecule generation through protein engineering, and selection of binders from large libraries of affinity molecules through techniques such as phage display, synthetic peptide libraries, or yeast display (Bradbury et al 2011, McCafferty and Schofield, 2015). Yeast display of single chain fragment variable (scFv) antibodies is used to generate binders specific to biomarkers of interest (Feldhaus et al 2003) with applications for diagnostics (Venkatesh et al 2015). The ability to use flow cytometry for selection, as well as for the verification of the functionality for the selected yeast displayed scFvs, makes this a very attractive approach to identify binders with the desired affinity properties (Feldhaus et al 2003; Gray et al 2010) (Figure 1A).…”

An Audio Jack-Based Electrochemical Impedance Spectroscopy Sensor for Point-of-Care Diagnostics

“…Glass slides sputtered with 10 nm Cr and 200 nm gold were used for the working and reference electrodes due to their superior impedance spectra in the appropriate frequency range compared to that of screen printed electrodes [25]. The sputtered glass slide with continuous gold film was segmented with a diamond-tipped pen to pattern two WEs and one common RE as shown in Fig.…”

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

“…24 More recently, whole yeast cells expressing scFv on their surface were modified with gold binding peptide, to allow a simple and cost-effective method for conjugation of the whole yeast cell sensor to a gold substrate. 30 …”

“…18−23 This approach of stabilizing surface proteins in a native environment has been adapted to stabilize recombinant antibody fragments from yeast display libraries. 24−30 ScFvs are stabilized by maintaining the native cell wall environment of the yeast display library from which they were selected. The yeast cell wall is comprised of a semirigid polysaccharide/lipid/protein matrix, which provides structure and rigidity to the cell, protection against physical stress, and a scaffold for surface proteins.…”

“…Whole yeast–scFv: scFv displayed on yeast cell surfaces are kept attached to the cell wall during immunoassays, hence maintaining the environment for which they were selected. Reprinted with permission from ref (30). Copyright 2015 Elsevier.…”