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Carbon-Nanotube Protein Microarrays Yield Sharp Improvement in Sensitivity

A team of researchers have developed a protein microarray technology linking surface-enhanced Raman scattering with single-walled carbon nanotubes that they said improves detection by three orders of magnitude over standard fluorescence-based assay methods.
Described in an article published Oct. 26 in the online edition of Nature Biotechnology, the technology is designed to increase the sensitivity and specificity of protein arrays for research and clinical applications.
In the article, the authors said that among the different technologies used for protein identification for clinical purposes, protein microarrays are among the most common because they provide “high-throughput, multiplexed protein detection for a range of applications.”
However, the sensitivity of such arrays, which are based on fluorophore tags, is limited to only about 1 picamole due to background interference or autofluorescence resulting from assay reagents or materials. Increasing the sensitivity, they add, would improve their usefulness in proteomics research.
At the same time, the explosion in biomarker-discovery work has created the need for a platform with greater sensitivity and specificity “to facilitate minimally invasive risk assessment, early-stage disease diagnosis, and [the ability to monitor] responses to therapeutic interventions,” the authors wrote in their article.
In addition to improving sensitivity and specificity, expanding the dynamic ranges of array technologies would benefit proteomics researchers by allowing researchers to compare more samples simultaneously, thus increasing throughput while reducing reagent use.
Two approaches currently being developed by other researchers — label-free, nanowire-based transistors and amplified detection based on multifunctional nanoparticles — have shown promise but also have significant limitations, the researchers said.
Label-free nanowire-based transistors are sensitive in the fentomole range but only in samples in pure water or low-salt solutions, meaning that such sensitivity cannot be achieved in serum or other biological fluids.
Meanwhile, amplified detection based on multifunctional nanoparticles is sensitive beyond the fentomole level but requires multiple reagents, and is time consuming, according to the authors.

“Application of background-free, surface-enhanced multicolor SWNT Raman labels may eventually enable simultaneous detection of multiple analytes in complex fluids, with 1-fM sensitivity in a multiplexed, arrayed fashion.”

In contrast, the technology they’ve developed “has simpler requirements, may be easily multiplexed, and exhibits high sensitivity in clinically relevant samples over the [nanomolar] to [fentomolar] range,” they wrote.
Their technology builds on existing methods. Researchers have used surface-enhanced Raman scattering, or SERS, to detect immobilized proteins by coupling Raman-active dyes to gold nanoparticles functionalized by ligands. However, the weak intensities of “typical” Raman labels have limited the utility of such sensors: Sensitivity is not quantitative or is limited to the nanomolar range, short of what can be achieved with fluorescence methods.
Single-walled carbon nanotubes, or SWNTs, however, are “ideal labels for SERS-based protein detection,” according to the researchers. They have distinct electrical and spectroscopic properties, including strong and simple resonance Raman signatures. They also “possess enormous Raman scattering cross-sections … have simple and tunable spectra, and are more stable than other organic Raman labels,” they said.
While carbon nanotubes have been used as in vivo and in vitro optical probes for biological imaging, “their potential as Raman tags for highly sensitive detection applications has not been explored,” they added.
The work described in Nature Biotechnology seeks to change this.
They begin with highly water-soluble macromolecular SWNT’s functionalized with PEGylated phospholipids, and conjugated them with a secondary antibody, goat anti-mouse immunoglobulin G. They then developed a six-arm, branched, carboxylate-terminated PEG. Grafted onto gold-coated surfaces for protein immobilization, “this afforded excellent protein attachment and substantially helped to overcome non-specific binding,” the researchers said.
They enhanced Raman scattering intensity by annealing the gold-coated substrate in a reducing hydrogen atmosphere to aggregate the gold film into particles. A 5-nanometer layer of pure silver was also deposited onto the assay layer. “Both techniques reproducibly enhanced SWNT Raman signal [about] 60-fold without damaging SWNTs,” the authors said.
In order to increase the specificity of their technology, they functionalized the SWNTs with a 1:1 mole ratio mixture of 1,2-distearoyl-sn,glycero-3-phosphoethanolamine-coupled branched-methoxyPEG, and a linear 1,2-distearoyl-sn-glycero-3-phospohethanolomine carbamyl-PEG-amine to provide sites for bioconjugation.
They then compared their technology against standard fluorescence-based protein microarrays for the detection of anti-human serum albumin. For the protein microarrays, the background noise of substrates and reagent molecules sensitivity to about 1 picamole.
Their arrays, however, had less background noise and an improved signal-to-noise ratio “resulting from bright Raman scattering spectra and surface-enhanced techniques” and provided a broader dynamic range than fluorescence-based techniques. Their arrays, they said, improved sensitivity 1,000-fold over fluorescence methods over seven to eight orders of magnitude in dynamic range for systems with high-affinity interactions, they said.
They also tested their technology on anti-proteinase 3, a biomarker for the autoimmune disorder Wegener’s granulomatosis, and found it was able to detect aPR3 diluted up to 107-fold in 1 percent human serum.
While their technology allows for a 1,000-fold improvement in protein detection over fluorescence-based techniques, the authors said that sensitivity is dependent on a number of factors: Individual SWNT Raman tags bound to the same analyte will show a varying range of scattering enhancement factors due to the location of the nanotubes relative to metal structures, the local field enhancement of SERS hot spots, and the spatial and statistical distribution of the hot spots, they said, adding analyte concentration will also affect sensitivity.
Finally, they said that though they analyzed antibody-antigen interactions, their technology should be equally applicable for specific biomolecule targeting and can be used to probe protein-protein interactions and nucleic-acid hybridization.

“Application of background-free, surface-enhanced multicolor SWNT Raman labels may eventually enable simultaneous detection of multiple analytes in complex fluids, with 1-fM sensitivity in a multiplexed, arrayed fashion,” they said.

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