Development of a “Membrane Cloaking” Method for Amperometric Enzyme Immunoassay and Surface Plasmon Resonance Analysis of Proteins in Serum Samples

Abstract

Detection of trace amounts of target proteins in the presence of high concentrations of matrix proteins (e.g., serum samples) without separation steps is of great significance to biomedical research but remains technically challenging. Here we report a "membrane cloaking" method to overcome nonspecific protein adsorption and fouling problems for label-free surface plasmon resonance detection and heterogeneous immunosensing. A thin, hybrid, self-assembled monolayer on gold was formed with 70 mol % mercaptopropanol and 30 mol % cysteamine/propanedithiol to facilitate membrane fusion and covalent attachment of antibodies. After antibody immobilization, the surface was incubated with lipid vesicles, which fused to form a supported membrane. The analyte spiked in serum was introduced for binding, and the membrane and nonspecifically adsorbed proteins on the membrane were subsequently removed using a nonionic surfactant before the final measurement was carried out. Selection of a suitable surfactant can preserve antibody/antigen binding and selectively remove the membrane, allowing accurate measurement of the captured proteins without interference from nonspecifically adsorbed species. Surface plasmon resonance (SPR) quantification of IgG spiked in undiluted serum ( approximately 75 mg/mL protein) was achieved with the membrane cloaking method, whereas direct measurement without membrane removal resulted in a significantly large error. The cloaking method was also used to develop an enzyme amplified amperometric assay using HRP-conjugated IgG. Detection of concentrations as low as 5 fM proteins was obtained. Finally, a membrane cloaking assay combining SPR and in situ electrochemical measurement was demonstrated on a gold substrate. Similar sensitivity was observed using a continuous flow injection measurement. The method opens new avenues to develop direct assay methods with ultrahigh sensitivity for protein samples using SPR and enzyme-linked amplification mechanisms.

Monolayers of 3-Mercaptopropyl-amino Acid to Reduce the Nonspecific Adsorption of Serum Proteins on the Surface of Biosensors

“…26 A membranecloaking methodology was recently developed to remove nonspecific adsorption after a sample was exposed to biological fluid, without disrupting the biomarker bound to the molecular receptor. 27 In spite of these results, the mechanism of protein adsorption is not clearly understood. 28 It is suggested that different proteins consecutively adsorb at surfaces, starting with the most prominent protein in serum, albumin, followed by larger proteins such as IgG and fibrinogen.…”

Bioinspired assemblies and plasmonic interfaces for electrochemical biosensing

“…A similar experimental setup was used to monitor target DNA recognition using SPR and ACV concurrently, which allowed for optimal nucleic acid probe sequence selection from the optical and electrochemical data [106]. Cross-platform protein recognition, important in the field of immunosorbent assays (ELISA), has been successful via a “membrane cloaking” method that was developed for complementary results to be obtained by SPR and amperometric techniques [107]. Once antibodies were immobilized over a self-assembled monolayer, a cloaking phospholipid membrane was applied to the surface, which was resistant to nonspecific binding and could be removed once the assay was complete.…”

Nanobiosensors and Nanobioanalyses

“…This strategy was employed for the detection of undiluted serum spiked with IgG (Phillips et al 2007 ). A membrane cloaking strategy was designed to remove nonspecifi cally bound proteins on an SPR sensor, leaving only the analyte bound to the molecular receptor at the surface.…”