Electrochemiluminescent immunosensor for detection of protein cancer biomarkers using carbon nanotube forests and [Ru-(bpy)3]2+-doped silica nanoparticles

Abstract

We report the first electrochemiluminescent immunosensor combining single-wall carbon nanotube forests with RuBPY-silica-secondary antibody nanoparticles for sensitive detection of cancer biomarker prostate specific antigen.The measurement of protein biomarkers holds significant promise for early cancer detection. 1 , 2 Detection of multiple protein biomarkers provides more reliable diagnostics than single biomarker measurements.1 -3 Proteins in serum can be measured with enzyme-linked immunosorbent assays (ELISA), 4 radioimmunoassay (RIA),5 electrophoretic immunoassay6 and mass spectrometry-based proteomics.7 However, there is a clear need for simple, rapid, sensitive, accurate and low cost protein detection suitable for point of care use.Electrochemiluminescence (ECL) is initiated when tris (2,2′-bipyridyl)ruthenium(II), [Ru-(bpy) 3 ] 2+ , or RuBPY, in its oxidized form reacts with a suitable sacrificial reductant, and has been used for detection of DNA and proteins. [8][9][10] Magnetic bead ECL methods have been commercialized. 11,12 Dye doped silica nanoparticles show promise as labels in ultrasensitive ECL bioassays. 13 RuBPY can be trapped inside mesoporous silica nanoparticles synthesized in water-in-oil (W/ O) microemulsions. 14 A single nanoparticle can encapsulate thousands of RuBPY to provide large signal amplification. Surfaces of the particles can be decorated with attached antibodies or proteins and arrays utilizing ECL can be fabricated using analytical spots on a simple conductive chip with a single connection to a power source and readout with a CCD camera, as we demonstrated with DNA-based toxicity screening arrays.9 This approach is simpler than the commercial bead-based ECL assays, which require sophisticated and expensive sample manipulation and measurement systems.15In this communication, we combine ECL nanoparticle labels with a single-wall carbon nanotube (SWCNT) forest platform in a sandwich immunoassay procedure for protein detection. The SWCNT forests feature self-assembled 20-30 nm long terminally carboxylated SWCNT standing in upright bundles on a thin Nafion-iron oxide layer on a pyrolytic graphite † Electronic supplementary information (ESI) available: Experimental details, Fig. S1 16 and provide a large conductive, functionalized surface area for attachment of capture antibodies in immunoassays.16b We have used SWCNT forests to build ultrasensitive amperometric immunosensors and detected PSA in serum using multiple enzyme labels.17 However, arrays using this strategy involve microfabricated multi-electrode chips as well as multi-electrode potentiostats, which are not necessary in ECL arrays.9In related work, [Ru-(bpy) 3 ] 2+ immobilized on composite films of disordered CNTs in combination with Nafion, 18a partially sulfonated polystyrene 18b and Eastman-AQ polymers 18c were used for ECL determination of tripropylamine 18 with a detection limit of 3 pM. 18c SWCNT forests with large carboxylated surface areas combined with immunoassays using RuBPY-silica-secondary ant...

Theory and Simulation for Optimising Electrogenerated Chemiluminescence from Tris(2,2′‐bipyridine)‐ruthenium(II)‐Doped Silica Nanoparticles and Tripropylamine

“…[10] This is particularly important to avoid undesired background emissions from biological analytes. [11][12][13] Secondly, the excitation system (electrode potential) is decoupled from the detection signal (light) and any potentially transferred electrical noise is filtered by diffusion of the organic reactant intermediate. This leads to very low levels of electrical perturbation of the ECL light intensity that is used as the analytical signal.…”

Biofunctionalization of Nanomaterials

“…A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on HRPdoped nanosilica particles with the conjugation of anti-IgG antibodies [74]. Coupled with SWNT forests, an immunosensor array has been constructed via water-in-oil (W/O) microemulsions for PSA detection, with a detection limit of 40 pg mL −1 [75]. The conjugated polymer-amplified silica NP-based immunoassay has been also constructed for IgG detection.…”

Electrochemical immunosensor for prostate-specific antigen using a glassy carbon electrode modified with a nanocomposite containing gold nanoparticles supported with starch-functionalized multi-walled carbon nanotubes

“…Since pristine carbon nanotubes (CNTs) are chemically inert, it is necessary to activate the graphitic surface of the nanotubes in order to combination of functional material with carbon nanotubes. CNTs are usually functionalized by harsh oxidative processes, such as refluxing in a mixture of HNO 3 andH 2 SO 4 to generate defects on the sidewalls and tube tips, those defects can serve as anchor groups for functionalization and/or can provide sites for the coordination chemistry [27][28][29]. However, due to the introduction of a large number of defects, such chemical oxidation methods reduce the electrical conductivity and corrosion resistance of CNTs.…”