Electrochemical immunoassay platform for high sensitivity protein detection based on redox-modified carbon nanotube labels

Abstract

We report a highly sensitive immunoassay protocol based on the use of redox-modified multi-walled carbon nanotubes (MWNTs) as electrochemical labels. The MWNTs were coated with methylene blue (MB) at an optically-determined loading of 3.41 × 10(-3) mol g(-1), and were then attached to secondary antibodies (Ab(2)) by adsorption. As a model analyte mouse IgG was collected by primary antibody (Ab(1))-coated magnetic beads. Following binding of the MB-MWNT-Ab(2) conjugates, IgG could be measured by MB reduction. Using differential pulse voltammetry for quantification, IgG was calibrated with a dynamic range of 0.1 pg mL(-1) to 100 pg mL(-1). Given the different possible Ab(1)-MB-MWNT-Ab(2) orientations on the magnetic beads, it was likely that not all the MB communicated with the electrode. A greater quantity of MB could be accessed by using the Fe(CN)(6)(3-/4-) redox couple as a solution phase mediator. This enabled us to lower the dynamic range down to 5 fg mL(-1) to 100 fg mL(-1).

Electrochemical Immunoassay for Salmonella Typhimurium Based on an Immuno‐magnetic Redox Label

“…This is approx. twice the value found previously for methylene blue on MWCNTs (3.41×10 −3 mol g −1 ), which is in rough agreement with the MWCNTs here possessing a smaller radius ( r ), thus leading to a surface/volume ratio (=2/ r ) approx. 2.5 to 5 times greater than the range of MWCNTs used in the previous case ((diameters 20–40 nm ).…”

“…Methylene blue/MWCNTs pellets were prepared according to the literature with some modifications . MWCNTs (50 mg) were dispersed in 50 mL of milliQ water and then sonicated for 2 h. This suspension was mixed with 50 mL of 1 mM methylene blue and then sonicated for a further 1 h to allow binding between MWCNTs and methylene blue.…”

“…However, similar to metal nanoparticles, redox mediators offer the advantage of having less stability considerations during long term storage than enzymes, and may provide high sensitivity if a high enough quantity of charge is delivered per binding event. The redox mediator methylene blue has been shown to adsorb strongly to carbon nanotubes and has been shown to be an effective immunoassay label in this configuration . Herein we describe a novel bi‐functional electrochemical label consisting of magnetic microparticles bearing both monoclonal anti‐ Salmonella antibodies and a high coverage of the redox mediator methylene blue.…”

Lab-on-chip systems for integrated bioanalyses

“…What is detected is either the fluorescence from the fluorophore, or the chemiluminescence or optical absorption produced by reactions of the enzymatic label. Many other labels have been proposed, such as magnetic nanoparticles [ 37 – 39 ] or redox-active molecules [ 40 – 44 ]. A main issue with label-free methods is that, in general, any non-specifically adsorbed species contributes to the signal, whereas labelled methods offer an additional step of specificity (at the cost of a more complex sequence of analysis steps and the use of more chemicals–with the corresponding increased cost and analysis time).…”

Highly sensitive electrochemical detection of DNA hybridisation by coupling the chemical reduction of a redox label to the electrode reaction of a solution phase mediator

“…We previously presented hydrodynamic linear sweep voltammograms in support of this interpretation, and used Fe(CN) 6 3to mediate the reduction of MB adsorbed onto carbon nanotubes, for an immunoassay label. 31 Here we examined the use of Fe(CN) 6 3-/4to mediate charge from the PSA-(PAA/PSS) 2 (MB/PSS) 3 spheres. We measured the direct and Fe(CN) who used a lipase label to cleave ferrocene derivatives bound to an electrode (20 aM).…”