Label-Free, Single Protein Detection on a Near-Infrared Fluorescent Single-Walled Carbon Nanotube/Protein Microarray Fabricated by Cell-Free Synthesis

Abstract

Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.

Chirality dependent corona phase molecular recognition of DNA-wrapped carbon nanotubes

“…Such capabilities open up new possibilities in the biomedical field by allowing researchers to study complex biological processes by monitoring several important bio-markers at the same time, using selective sensors with different chiralities. SWNT sensors were utilized for monitoring nitric oxide in vivo [13], vitamin dynamics within cells [5], and proteinprotein interactions [18]. In addition to multiplexed detection and improved signal to noise ratio, chirality specific sensors would allow for an internal fluorescent standard that would eliminate the need for signal calibration or allow for ratiometric sensing.…”

“…These properties, along with the capability of single molecule detection [16], have made SWNT nanosensors valuable tools to study physical, chemical and biological processes at short length and time scales otherwise not obtainable from other detection platforms. As a result, considerable effort has been devoted to the design and development of SWNT-based optical sensors for a variety of target analytes including neurotransmitters [17], proteins [18][19][20], carbohydrates [21][22][23] and assorted small molecules [5,16,24].…”

Protein functionalized carbon nanomaterials for biomedical applications

“…Earlier work demonstrated the capture of his-tagged proteins, expressed using cell-free synthesis, by a nickel chelation group on chitosan wrapped SWCNT sensors array, followed by the detection of a model analyte of anti-histag antibody (Fig. 3c) [106]. A follow up work demonstrated glycoprofiling using a similar platform, where his-tagged recombinant lectins were used as the capture protein, successfully detecting streptavidin-tethered biotinylated monosaccharides [111,112].…”

Chemical sensors based on polymer composites with carbon nanotubes and graphene: the role of the polymer

“…Other sensors involve a polymer that acts as a matrix 123,124 or immobilization support. 125,126 In this regard, Barone et al…”

“…As a proof of concept, these sensors were implanted into a mouse tissue, demonstrating an excellent signal-to-noise ratio of 8.6. Similarly, Ahn et al 124 prepared a sensor for protein detection based on SWCNTs embedded within a chitosan matrix bearing an NR,NR-bis(carboxymethyl)-L-lysine (NTA) chelator (Fig. 3).…”