Electrochemical immunosensors for detection of cancer protein biomarkers

Abstract

Bioanalytical methods have experienced unprecedented growth in recent years, driven in large part by the need for faster, more sensitive, more portable ("point of care") systems to detect protein biomarkers for clinical diagnosis. Electrochemical detection strategies, used in conjunction with immunosensors, offer advantages because they are fast, simple, and low cost. Recent developments in electrochemical immunosensors have significantly improved the sensitivity needed to detect low concentrations of biomarkers present in early stages of cancer. Moreover, the coupling of electrochemical devices with nanomaterials, such as gold nanoparticles, carbon nanotubes, magnetic particles, and quantum dots, offers multiplexing capability for simultaneous measurements of multiple cancer biomarkers. This review will discuss recent advances in the development of electrochemical immunosensors for the next generation of cancer diagnostics, with an emphasis on opportunities for further improvement in cancer diagnostics and treatment monitoring. Details will be given for strategies to increase sensitivity through multilabel amplification, coupled with high densities of capture molecules on sensor surfaces. Such sensors are capable of detecting a wide range of protein quantities, from nanogram to femtogram (depending on the protein biomarkers of interest), in a single sample.

Fig. 1

Scheme of the sandwich assay for protein biomarker detection using magnetic beads. Antibody-modified magnetic beads capture the protein from sample solution, followed by binding of a second enzyme-labelled antibody. Enzyme‘s substrate is used to develop the electrochemical signal. WE is the working electrode.

Fig. 2

Bioconjugates for signal amplification strategies in electrochemical immunosensors. After the capture antibody is immobilized on the sensor surface, and the analyte protein is captured, these bioconjugates bind with analyte in a sandwich immunoassay. An electrochemical signal is generated using a substrate suitable for the electroactive species (typically an enzyme) on the bioconjugate probe. (a) Ab₂-enzyme, (b)Ab₂-nanoparticle, (c) Ab₂-biotin-strepatavidin-enzyme, (d) Ab₂-CNT-enzyme, (e) CNT-(PDDA-AP)₄-PDDA-PSS tag, (f) multienzyme-Ab₂-nanoparticle, (g) Ab₂-nanoparticle-Qdots, (h) Ab₂-MB-multienzyme clusters, (i) MB-AuNP-Ab₂-multienzyme.

Fig. 3

Multiprotein detection protocols using multiple labels or multiple electrodes. (a) Magnetic beads coated with differing primary antibodies are used to capture their corresponding antigens. Then, secondary antibodies labelled with differing inorganic nanocrystal tracers bind to the corresponding antigens. Following acid dissolution of the nanocrystals, the resulting ions are detected by electrochemical stripping. Adapted with permission from reference. (b) Magnetic beads labelled with multi-enzymes and Ab₂ were used to capture specific analytes offline, and a sandwich immunoassay was performed on an 8-electrode array modified with different antibodies. (c) Multiplexed immunoassay using an array of screen printed carbon electrodes modified with different capture antibodies employing universal MWCNT bioconjugates. Adapted with permission from reference.

Fig. 4

Different sensor surfaces for sandwich immunoassays, usable with electrochemical detection. (a) Carbon fiber microelectrode, modified with AuNP and protein A to capture a high density of primary antibodies. Adapted with permission from reference. (b) SWCNT forest assembly developed on a pyrolytic graphite (PG) surface. Adapted with permission from reference. (c) AuNP-modified pyrolytic graphite surface using layer-by-layer (LBL) approach. Adapted with permission from reference. (d) Glassy carbon electrode modified with silver-MWCNT composite. Adapted with permission from reference. (e) SWCNT-modified Pt substrate used for label-free detection. Adapted with permission from reference.

Fig. 5

Different approaches for ECL immunosensors, with corresponding data and calibration curves. (a) ECL immunosensor employing Au-silica-CdSe-CdS QD nanostructure for the detection of CEA. Immunoassay developed on the sensor surface inhibits electron transfer and decreases ECL intensity. Adapted with permission from reference. (b) ECL system employing quenching of ECL upon immunocomplex formation. Adapted with permission from reference. (c) ECL immunosensor assembly employing GOx label as a coreactant to generate ECL. Adapted with permission from reference. (d) ECL immunosensor developed on SWCNT-modified pyrolytic graphite electrodes using silica-Ru(bpy)₃²⁺ particles. Adapted with permission from reference.

Fig. 6

Microfluidic devices employed for electrochemical detection. (a) Microfluidic device fabricated with PMMA and an 8 electrode array for simultaneous detection of biomarkers, employing offline capture of protein analytes. Unpublished photo by B.V.Chikkaveeraiah. (b) Paper-based microfluidic device for electrochemical detection. Adapted with permission from reference. (c) Paper based device for ECL sensor. Adapted with permission from reference. (d) 3D paper based ECL device with screen printed carbon working electrodes. The common Ag/AgCl reference and carbon counter electrodes are on another paper, and interface by stacking. Adapted with permission from reference. RE- reference electrode, CE- counter electrode, WE- working electrode.

Fig. 7

Paper-based microfluidic ECL sensor system. (a) Paper microfluidics produced in bulk using an inkjet printer. (b) The hydrophilic portion of the paper is filled with a 10 mM Ru(bpy)₃²⁺ solution and then dried. (c) The paper substrate is then laminated onto the screen-printed electrode using transparent plastic. A drop of sample is introduced through a small aperture in the plastic at the base of the channel. Then, after the detection zone is fully wetted, a potential of 1.25 V is applied, and the sensor is placed close to the lens of a camera phone (d) to capture the resulting emission. (e) ECL images from the paper-based sensor for various concentration of DBAE. (f) Calibration curve showing ECL response between 0.5 mM and 20 mM DBAE, using paper microfluidic ECL sensor. Adapted with permission from reference.

Scheme 1

Protein detection chemistry employing amperometry using HRP (PFe^III) labels. Iron heme enzyme (PFe^III) is oxidized to ferryl oxy species upon reaction with H₂O_2, and then is reduced by the mediator hydroquinone (H₂Q), resulting in the formation of water and quinone (Q). Quinone gets reduced at the electrode back to hydroquinone, giving rise to an electrical signal.

Scheme 2

Pathway for ECL generation in Ru(bpy)₃]²⁺/TPrA system. The ECL pathway involves generation of ECL by reduction of [Ru(bpy)₃]²⁺ to [Ru(bpy)₃]⁺ mediated by direct oxidation of TPrA on the sensor surface. Reproduced from reference.