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Xu Group at ODU Reports Major Advance in Creation of Photostable Single-Molecule Biosensors

Members of the research group of Old Dominion University scientist X. Nancy Xu report in the Journal of the American Chemical Society (JACS) that they have created extraordinarily tiny silver nanoparticles that can be used as biosensors to study molecular-level functions of a protein that mediates biological functions affecting human health.

"Photostable Single-Molecule Nanoparticle Optical Biosensors for Real-Time Sensing of Single Cytokine Molecules and Their Binding Reactions" was written by Xu, associate professor of chemistry and biochemistry, and by postdoctoral researcher Tao Huang and graduate student Prakash Nallathamby. The article appeared online in November and is scheduled to appear soon in the print version of JACS, the flagship journal of the ACS.

"This study represents a major advance in preparing photostable noble metal dots for imaging and sensing of low and high concentrations of proteins of interest and probing their interactions at the single molecule resolution, which has not yet been reported previously," Xu said.

The researchers state that their breakthroughs provide a new and improved way to investigate a pro-inflammatory cytokine that is called tumor necrosis factor-alpha (TNF-alpha). This regulatory protein can mediate a variety of biological effects, such as immune regulation, antitumor activity and infection resistance. When TNF-alpha regulation is out of whack, especially when it is overproduced, it can signal a pathological condition such as cancer, heart disease, diabetes and autoimmune disease.

"Unfortunately, despite extensive research over decades, the underlying mechanisms about how TNF-alpha mediates these crucial biological functions still remain incompletely understood," the researchers write. They add that it is therefore "important to develop ultrasensitive assays for accurate analysis of TNF-alpha."

Because it takes only a few cytokine molecules to induce a significant cellular response, very precise means are required to detect and image the molecules and to characterize their functions in real time. Many conventional detection methods, the researchers write, are time-consuming and cannot be used in quantitative analysis of TNF-alpha in real time.

The work also offers a powerful tool-the photostable probes-to study single molecule reactions in real time. Currently, single molecule detection (SMD), an ultrasensitive means that allows detection of individual molecules and counts one molecule at a time, is limited by the brief lifespan of fluorescence probes. Xu and her team have been working in recent years with silver nanoparticles about 12 nanometers in diameter to probe single protein molecules on living cells in real time, and they have found that unlike florescent probes, these nanoparticles do not suffer photodecomposition and can stay on the job for long periods.

Could these nanoparticles be the delicate, yet long-lasting probes needed to detect and study TNF-alpha molecules? Yes, but with some modifications, the researchers found. For their TNF-alpha investigations, the researchers needed to synthesize smaller silver nanoparticles-about 2.5 nanometers in diameter-that were the basis of more useful sensors. These nanoparticles are so small (only about 430 atoms in total) that their surface area is greater than their bulk volume. Thus, surface properties of individual nanoparticles pay key roles in defining their physical and chemical properties. Consequently, optical properties of individual nanoparticles are extremely sensitive to the change of their surface properties, which forms the basis of design of such sensitive, single-molecule nanoparticle biosensors.

To the tiny nanoparticles, the researchers affix single monoclonal antibody (MAB) molecules to create single molecule nanoparticle optical biosensors (SMNOBS) that attract TNF-alpha molecules. Dark-field single nanoparticle microscopy and spectroscopy, developed by the Xu group, enables the sensing and detection of single protein molecules, which are in effect "switching the color" of single nanoparticles. "We found that the SMNOBS resisted photodecomposition and could be used for imaging and quantitative analysis of single protein molecules (TNF-alpha) and its binding reactions for hours," the researchers wrote.

The researchers also assert that "intrinsically superior features" allow their SMNOBS to detect and sense proteins of interest in low and high concentrations at the single molecule resolution, offering a large dynamic range. The biosensors are "well suited both for diagnosis of TNF-alpha-related diseases and for probing their fundamental roles in a variety of biological functions," according to the authors. "The tiny sizes of SMNOBS also offer the possibility of them being delivered into living organisms for sensing biomolecules of interest and probing their function in small organelles in real time."

Earlier this year, the Xu group reported in the American Chemical Society journal, ACS Nano, its discovery of new ways to synthesize silver nanoparticles that are particularly well suited for complex probes of live zebra fish embryos. New monodisperse-or size-consistent-nanoparticles that the researchers create using a simple washing step are stable (non-aggregated) in solution for months.

The group's findings solve a major obstacle-stability of nanoparticles in solution-for achieving a wide variety of applications of nanoparticle probes, such as an in vivo imaging. The researchers deploy these tiny probes to simultaneously image multiple nanoenvironments of in vivo biological systems, such as zebra fish embryos, in real time.

The June 2008 article, "Design of Stable and Uniform Single Nanoparticle Photonics for In Vivo Dynamics Imaging of Nanoenvironments of Zebra Fish Embryonic Fluids," was written by Xu and Nallathamby, as well as Kerry Lee, a doctoral student in the Xu group.

In 2007, the Xu group reported the first results of their study of the entry of nanoparticles into zebrafish embryos and of dose-dependent toxicity of silver nanoparticles on embryonic development. One aim of Xu's research is the development of an in vivo system to screen the biocompatibility and toxicity of nanomaterials.

Single-nanoparticle photonics and the single nanoparticle imaging system developed by the Xu group enable the direct characterization of size and location of nanoparticles inside cells and embryos.

Beginning about a decade ago, Xu's research put her in the vanguard of scientists using very tiny nanoparticle optical sensors to study living cells. She currently has grants totaling more than $2.5 million from the National Science Foundation and the National Institutes of Health to support her work. She is the recipient of a 2008 Nanotech Briefs Nano 50™ Innovator Award and 2007 Nanotech Briefs Nano 50™ Technology Award.

Xu was named in a 2006 article prepared by the National Cancer Institute as a pioneering developer of nanotechnology that can be used in the war against cancer. The article, titled "Mission to the Inside of a Living Cell," noted the benefits of studying biochemical reactions inside live cells. Similar studies in the past were conducted with dead cells or purified biomolecules extracted from cells. The article also gave the Xu group high marks for producing silver nanoparticles that are exceedingly bright and do not photodecompose.

This article was posted on: December 3, 2008

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