(100e) Label-Free QCM Immunobiosensor for Real-Time Detection of GFP Antigen Using IOS-1 Peptoid:

The medical diagnostic industry is in need of cheaper, time-saving, and more robust assays for pathogen detection. Our research approach is focused on the development of short-chain designer peptoids that can be tuned to target detection of specific biological molecules. The need for detection of specific pathogens has necessitated our design of a designer peptoid (IOS-1), which preferentially isolates a target antigen (i.e., pathogen or pathogen metabolite) from a dynamic aqueous stream. When incorporated into diagnostic tools, these designer peptoids, which are customizable to the extracellular matrix (envelope) protein of target pathogens, may proffer a cost-effective and rapid means of pathogen identification and quantification. In this research, the precise Quartz Crystal Microbalance (QCM) is used to track adsorption events in real-time.

In this work, we have developed a technique for the in situ detection of green fluorescent protein (GFP) antigen in flowing PBS buffer solution using an immunobiosensor comprising a gold substrate whose surface is modified ex-situ with IOS-1 peptoid. This peptoid is an oligomer of N-substituted glycines that was designed using the submonomer solid-phase synthesis approach.

The QCM provides real-time frequency shift data for the mounted immunobiosensor during the flow of analyte solution, baseline solution, and reflux solution. By using the Sauerbrey model, mass loading of the adsorbed analyte was calculated, thereby enabling in situ quantification of adsorbed GFP antigen. This immunobiosensor is robust enough for dynamic operation and is reusable in vitro by switching the flow of antigen with 0.1% sodium dodecyl sulfate solution to recondition the immunobiosensor surface before resumed capture of antigen. Equilibrium mass loadings of antigen before and after SDS reflux point toward reproducible surface chemistry for this immunobiosensor. Experimental data obtained using this setup are contrasted with data from a similarly constructed immunosensor consisting of the gold substrate which is surface modified with aminoethane thiol, disuccinimidyl suberate and GFP antibody in a stepwise immobilization scheme.

The average mass loading of peptoid immobilized on the (0.78 cm²) sensor surface was about 400 nanograms, and this peptoid monolayer can capture between 1 nanogram of the analyte and 200 nanograms of the analyte, depending on the initial concentration of antigen flowed across the immunobiosensor. Characterization of the surface was achieved using FTIR, fluorescence microscopy and XPS. Baseline QCM readings were taken in PBS buffer.