Carbon Nanotube Amplification Strategies for Highly Sensitive Immunodetection of Cancer Biomarkers

Abstract

We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab 2 ) linked to carbon nanotubes (CNT) at high HRP/Ab 2 ratio. This approach provided a detection limit of 4 pg mL −1 (100 amol mL −1 ), for prostate specific antigen (PSA) in 10 μL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.

Carbon Nanotubes for Electronic and Electrochemical Detection of Biomolecules

“…[27,112] This has propelled carbon nanotubes into novel and highly sensitive bioamplification strategies. [30,31] Another area in which bioelectrochemistry has made excellent progress is in DNA hybridization sensors for diagnosing genetic diseases. [113][114][115][116] As shown below, carbon nanotubes were similarly used to advance this area as well.…”

“…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.…”

“…[134] Our group has evaluated a number of SWNT forest platforms for amperometric protein immunoassays. [31,75] Figures 3b 12 and b 13 illustrate single-and multilabel detection strategies using HRP as electroactive label, where the electrode (shown as thick black line) consists of SWNT forest array (Figs. 3b 3 ).…”

“…This method allows binding and detection on a single surface, with straightforward washing and reagent addition (e.g., labeled antibody) steps performed via dipping or flow modes. Based on this approach, sensitive immunosensors for human serum albumin [75] and prostate specific antigen [31] have been developed, as described below.…”

“…[26] The facile electron transfer between nanotubes and a variety of protein redox cofactors, [27] in conjunction with conductive polymers [28,29] and nanoparticles [30] of metallic, semiconducting, and magnetic nature provide the means to implement new biosensing schemes and enhance the sensitivity of current methodologies. [27,30,31] CNTs can be readily dispersed as individual [32] and lightly bundled nanotubes [33,34] and assembled, [35] screen-printed, [36] and potentially inkjet-printed to produce device configurations with controlled transparency. This is further amplified by an assortment of covalent and noncovalent functionalization strategies to link a variety of biological entities onto CNTs.…”