Mediated amperometric immunosensing using single walled carbon nanotube forests

Abstract

A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes called SWNT forests. The forests were selfassembled from oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes. The nanotube forests were characterized using atomic force microscopy and resonance Raman spectroscopy. Anti-biotin antibody strongly adsorbed to the SWNT forests. In the presence of a soluble mediator, the detection limit for horseradish peroxidase (HRP) labeled biotin was 2.5 pmol ml 21 (2.5 nM). Unlabelled biotin was detected in a competitive approach with a detection limit of 16 nmol ml 21 (16 mM) and a relative standard deviation of 12%. The immunosensor showed low non-specific adsorption of biotin-HRP (approx. 0.1%) when blocked with bovine serum albumin. This immunosensing approach using high surface area, patternable, conductive SWNT assemblies may eventually prove useful for nano-biosensing arrays.

Carbon Nanotubes for Electronic and Electrochemical Detection of Biomolecules

“…Carbodiimide-based covalent linking of SWNTs to amine-functionalized substrates appears to decrease the SWNT forest coverage by one to two orders of magnitude as compared to the Fe 3+ -assisted electrostatic assembly. Both methodologies have provided significant advances for electrochemical biosensors through: i) Ease of fabrication using straightforward dipping and washing steps; [78] ii) Flexible patterning schemes; [122] iii) Availability of carboxy-functionality for convenient conjugation with a variety of biomolecules; [31,35,75,124] iv) Convenient SWNT length-fractionation to tune forest height and its resistivity; [123] v) Efficient vectorial electron transfer along the nanotube length with nearby cofactors of enzymes; [35,75] and vi) Excellent hydrodynamic stability at spinning speeds in excess of several thousand rpm. [35] On the other hand, the as-grown single-and multi-walled nanotubes, using chemical vapor deposition (CVD), display much lower surface coverage (10 7 −10 9 tubes cm −2 ) and upon drying they may collapse.…”

“…With the electroactive enzyme tag (i.e., HRP or ALP) catalyzing the redox conversion of a substrate (i.e., H 2 O 2 or α-naphthylphosphate, respectively), the signal produced is proportional to the number of enzyme tags at a fixed substrate concentration. [124] Assuming that the sensing antigen (Ag) has the same binding constant with the electro-active Ag-HRP, the signal reduction from the displacement of Ag-HRP by Ag will be proportional to the concentration of Ag. [134] Competitive assays inherently lack good detection limits owing to the difficulty of accurately measuring the difference between two large signals near the detection limit.…”

“…[124] Using hydroquinone (H 2 Q) as a soluble mediator, a detection limit of 2.5 pmol mL −1 was reached for HRP-labeled biotin.…”

Functionalized carbon nanotubes and nanofibers for biosensing applications

“…Rusling reported amperometric enzyme-linked immunoassays of proteins based on a vertically-aligned array of SWCNT forests [48][49][50]. A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly-oriented assemblies of SWCNT forests [48].…”

Trends and Perspectives in Immunosensors

“…An electrochemical immunosensor using a mediator and single walled carbon nanotube forests as the electrode surface was demonstrated in a competitive immunoassay for detecting biotin-HRP and unlabelled biotin. In this competitive assay, labelled and unlabelled antigens competed for binding sites on the anti-biotin capture Ab [42]. The mediator chosen for this system was hydroquinone, used to facilitate a catalytic reduction of hydrogen peroxide by transferring electrons between HRP and the peroxide.…”