Label‐Free Colorimetric Detection of Single Nucleotide Polymorphism by Using Single‐Walled Carbon Nanotube Intrinsic Peroxidase‐Like Activity

Abstract

Single-walled carbon nanotubes (SWNTs) have been considered to be leading candidates for nanodevice applications and novel drug delivery. Intriguingly, recent studies have shown that SWNTs have catalytic activity even in the absence of catalytic factors. Since hydrogen peroxide is an important oxidizing agent in biological systems, the catalytic reaction of SWNTs with H2O2 has received much attention. The SWNT catalytic mechanism is much debated, but it has been suggested that it is related to trace amounts of metal catalyst in SWNTs. H2O2 is a major reactive oxygen species in living organisms, and its overproduction is implicated in the development of numerous inflammatory diseases, such as atherosclerosis, chronic obstructive pulmonary disease, and hepatitis. Furthermore, as a product of many enzyme-catalyzed reactions, H2O2 can act as an indicator to monitor the quantity of biologically important molecules, such as glucose. Therefore, studying the catalytic reaction of SWNTs with H2O2 might offer a promising application for disease diagnosis and for the design of SWNT-based sensors. In this work, we report that SWNTs possess intrinsic peroxidase-like activity. That the catalytic activity does not depend on trace amounts of metal catalyst in the SWNTs is evidenced by energy-dispersive X-ray (EDX) analysis. In the presence of H2O2, SWNTs catalyze the reaction of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) thereby producing a color change (Scheme 1). Our results indicate that the catalytic efficiency of SWNTs is strongly dependent on pH, temperature, and H2O2 concentration, similar to horseradish peroxidase (HRP). More importantly, we compared the catalytic efficiency of SWNTs containing different amounts of cobalt residues. The results clearly show that the observed “catalytic” effect of SWNTs can be attributed to their intrinsic properties rather than metal residues. Peroxidase activity has a great potential for practical application and has been used in the bioremediation of waste water or as diagnostic kits. As peroxidase mimics, SWNTs were used here for label-free colorimetric detection of disease-associated single-nucleotide polymorphism (SNP) with a direct detection limit of 1 nm based on the color reaction of TMB. It is well known that SNP detection is very important, and different kinds of detection methods have been reported; however, to our knowledge, this is the first demonstration of applying intrinsic SWNT peroxidase-like activity and color change for this purpose. This work will provide new insights into the utilization of SWNT peroxidase-like activity. To increase SWNT solubility in aqueous solution, we treated SWNTs with a mixture of concentrated sulfuric and nitric acids, as described previously. Figure S1 in the Supporting Information shows the mixed solution of H2O2 and TMB in the presence or absence of SWNTs. In the absence of SWNTs, the color of the solution does not change in 12 h; however, in the presence of SWNTs, the color changes from black to blue immediately. This result suggests that SWNTs can catalyze the reaction of TMB in the presence of [a] Y. Song, X. Wang, C. Zhao, K. Qu, Dr. J. Ren, Prof. X. Qu Laboratory of Chemical Biology Division of Biological Inorganic Chemistry State Key Laboratory of Rare Earth Resource Utilization Graduate School of the Chinese Academy of Sciences Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun, Jilin 130022 (China) Fax: (+86)431-85262656 E-mail : xqu@ciac.jl.cn Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.20090902643. Scheme 1. Schematic illustration of SWNTs catalyzing the reaction of peroxidase substrate TMB in the presence of H2O2 to give the blue product oxidized TMB (oxTMB).