Nanomaterial Labels for Signal Amplification in Electrochemical Immunosensors
Authors: Manickam Pandiaraj , Bhansali Shekhar , Pasha Khalid , Huanghan Hui 
Published: February 2020

Electrochemical immunosensors are functions by measuring the changes in the electrical properties such as current, potential, conductance, capacitance, and impedance during an immunochemical reaction between antigen (target molecule) and the antibody(Pandiaraj et al., 2014; Vasudev et al., 2013). Although capacitive and impedimetric immunosensors can directly be utilized to investigate the antibody-antigen interaction without the need of other reagents and separation steps, their analytical sensitivity is limited in clinical applications. Amperometric immunosensors are based on the measurement of currents resulting from the electrochemical oxidation or reduction of electroactive species had attracted more interests recently for a higher sensitivity and less complicated instrumentation (Rusling et al., 2010). However, amperometric immunosensors requires labelling of either antigen or antibody, since both the reaction partners are electrochemically inert. Labelling is a time consuming process, costly and denatures the biomolecules. Amperometric immunosensors that utilizes electrolyte solution containing reversible redox species, such as ferrocene and its derivatives, dyes, hydroquinone, hexacyanoferrates etc. have been widely used as mediators or signal generating probes instead of labelling antibodies/antigens; however this would cause electrode contamination, reagent-consumption and operation inconvenience. To solve these problems, it should be preferable to immobilize the signal generating probe on electrode surfaces to construct label-free amperometric immunosensors.

The use of nanomaterials as signal generating probes for electrochemical detection of biomarkers have been reviewed earlier (Liu and Lin, 2007). However, the selection of signal generating probe is very important for electrochemical immunosensor, especially for label-free electrochemical immunosensor, since it is responsible for three major tasks: (i) being redox active and produce electrochemical signals for corresponding immunocomplex formation, (ii) increasing specific surface area of electrode to immobilize more antibodies and improving conductivity for sensitive detection of antigen. For electrochemical devices, nanoparticles, carbon nanotubes and graphene have been used to catalyze electrochemical reactions and to improve electron transfer. Nanoparticles, especially have also been used as labels to generate intense signal in comparison with electroactive molecules or organometallic complexes like ferrocene. Indeed, due to their high number of atoms, huge number of electron can be exchanged through oxidation or reduction.

Group 11 metals (Cu, Ag, and Au), which are very weak chemisorbers, often display marked catalytic and electrocatalytic properties. Some types of copper electrodes showed an excellent electrocatalytic activity for DNA detection without observable self-poisoning. Copper electrodes undergo oxidation at unexpectedly low potentials, typically within the so-called double layer region, to form surface-bonded copper(I) hydrous oxide species.

A reagentless, label-free voltammetric immunosensing strategy was demonstrated by employing nanomaterial integrated electrode as a signal generating probe. Copper/copper oxide (Cu/CuxO) nanoparticles integrated screen printed carbon electrodes (SPCE) were used to construct the immunosensing platform. The stress biomarker, cortisol was employed as a model analyte. Cortisol specific monoclonal antibodies (C-Mab) were covalently immobilized on the surface of the Cu/CuxO-SPCE functionalized with dithiobis(succinimidyl propionate) (DTSP) self-assembled monolayer (SAM). The formation of Cu/CuxO nanoparticles on the surface of SPCE was characterized by scanning electron microscopy (SEM), and X-ray diffraction (XDR) analysis. The redox signal of the Cu/CuxO nanoparticles could be detected directly through cyclic voltammetry and used as signal generation probe for the direct detection of cortisol. The formation of immunocomplexes leads to a decrease in the electrochemical redox signal of Cu/CuxO nanoparticles owing to increased spatial blocking of the electrode surface. The proposed immunosensing strategy allows a rapid and sensitive means of cortisol analysis with a limit of detection of about 1 pg/mL. The Cu/CuxO platform can be further applied for designing other label-free immunoassays.

References:

Liu, G., Lin, Y., 2007. Nanomaterial labels in electrochemical immunosensors and immunoassays. Talanta 74, 308–317. doi:10.1016/j.talanta.2007.10.014

Pandiaraj, M., Sethy, N.K., Bhargava, K., Kameswararao, V., Karunakaran, C., 2014. Designing label-free electrochemical immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes. Biosens. Bioelectron. 54, 115–21. doi:10.1016/j.bios.2013.10.030

Rusling, J.F., Kumar, C. V, Gutkind, J.S., Patel, V., 2010. Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer. Analyst 135, 2496–511. doi:10.1039/c0an00204f

Vasudev, A., Kaushik, A., Bhansali, S., 2013. Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection. Biosens. Bioelectron. 39, 300–5. doi:10.1016/j.bios.2012.06.012