An Ultrasensitive Biosensor for Detection of Femtogram Levels of the Cancer Antigen AGR2 Using Monoclonal Antibody Modified Screen-Printed Gold Electrodes.

Wioleta Białobrzeska,Radovan Krejcir,Katarzyna Pala,Zofia Cebula,Robert O’Neill,Borivoj Vojtesek,Ewelina Bięga,Natalia Malinowska,Daniel Bigus,Sabina Żołędowska,Dawid Nidzworski,Małgorzata Lisowska,Petr Muller,Karolina Dziąbowska,M Aiman Mohtar,Elżbieta Czaczyk,Ted R Hupp

doi:10.3390/bios11060184

Abstract

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

Highlights

Anterior Gradient-2 (AGR2) was identified as a key protein involved in the assembly of the dorso–anterior ectoderm that forms the cement gland and maintains forebrain integrity [1,2]
We aimed to use a gold electrode platform [33] to determine whether the AGR2 antigen can be detected in a ‘one-step’ process from aqueous samples using the monoclonal antibody validated in clinical tissue using immunohistochemistry [15] and in a two-site ELISA assay [31]
We have reported the extended investigation of an impedimetric immunosensor for AGR2 protein detection

Summary

Introduction

Anterior Gradient-2 (AGR2) was identified as a key protein involved in the assembly of the dorso–anterior ectoderm that forms the cement gland and maintains forebrain integrity [1,2]. AGR2 is over-expressed in a diverse set of human cancers including breast [11], prostate [12], pancreatic [13], liver [14], ovarian [15], esophagus [16,17,18], and lung cancers [19]. Mass spectrometry and ELISA methodologies have been used to detect AGR2 peptide fragments in urine or plasma [22,23,24,25,26]. The paper shows significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were elevated in patients with both serous and non-serous tumors. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients

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