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

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.

Electroanalytical overview: screen-printed electrochemical sensing platforms for the detection of vital cardiac, cancer and inflammatory biomarkers

“…In terms of alternative markers for other cancers very similar strategies of biosensor development to the ones highlighted for HER2 have been used. For example, there have been reports for the determination of miRNA-155 (a specific microRNA (non-coding RNA) shown to have diagnostic value in several cancer types including prostate, lung, squamous cell carcinoma and, most notably breast), 133 arginase-2 (AGR2) (an enzyme, which when overexpressed in many cancer types is responsible for the destruction of l -arginine and hence creates a localised immunosuppressive state) 134 using an Au-SPE modified with their biorecognition elements through Au–S bonds 135,136 and for prostate specific antigen (PSA) (one of the most well established cancer biomarkers for the diagnosis, treatment and follow-up of prostate cancer cases) using a simple antibody sandwich assay on C-SPEs, 137 aptamers deposited on AuNPs 138 and detection systems combining these two methods. 139,140 This latter example is reported for PSA initially, then followed up for the detection of both PSA and interleukin-6 (IL-6), a commonly used biomarker for many conditions that will be discussed in more depth in the latter section.…”

Porous Silicon-Based Aptasensors: Toward Cancer Protein Biomarker Detection

“…To further improve the detection reliability, the Morlet wavelet phase signal processing technique was applied instead of RIFTS and resulted in a substantial reduction in the measurement noise, which reduced the LOD to 0.66 μg mL –1 (15 nM). We acknowledge that the current LOD is not sufficient for AGR2 detection in real clinical samples and is inferior compared to other reported biosensors for AGR2 detection. − However, our constructed biosensor presents a superior simplicity and selectivity in a highly complex media, as well as direct AGR2 detection with a real-time binding resolution. We also argue that the LOD can be improved by the additional means of nanostructure optimization in terms of its thickness, introducing flow into the system for convection, the optimization of the microfluidic architecture, applying active or passive mixing approaches (e.g., microfluidic mixers or magnetic mixers), , and tuning the filtering parameters of the Morlet wavelet phase method.…”

“…20,21 Recently, a highly sensitive detection of AGR2 was demonstrated with an electrochemical immunosensor; however, selectivity was not shown in a clinically relevant medium. 22 In this work we aim to develop a label-free biosensor for the direct detection of the protein AGR2 in body fluids. The biosensor is constructed from porous silicon (PSi) Fabry− Peŕot thin films, which enable target detection via real-time monitoring of the PSi reflectance changes.…”

“…To date, AGR2 detection has been demonstrated by traditional laboratory-based analytical methods, such as enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS), which are time-consuming, laborious, and expensive. Aptamer-based optical biosensors (aptasensors) for sensitive AGR2 detection have been successfully constructed, relying on rather complex competitive reaction schemes. , Recently, a highly sensitive detection of AGR2 was demonstrated with an electrochemical immunosensor; however, selectivity was not shown in a clinically relevant medium …”

Recent advances in gold electrode fabrication for low-resource setting biosensing

“… 33 Bialobrzeska et al , used the Dropsens BT electrodes to detect the cancer antigen AGR2 in lysed cell samples with a LOD of 0.093 fg mL −1 and a linear range of 0.001 fg mL −1 to 0.900 fg mL −1 . 34 These examples shed light on the potential of SPEs to be used in biosensor fabrication.…”