Ian D. Walton

Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R2 = 0.998) or i.v. (R2 = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.

Electrochemical synthesis and optical readout of striped metal rods with submicron features

Abstract Fluorescent molecules are widely used as identification tags in both analytical and bioanalytical chemistry. This manuscript describes an electrochemical approach to the manufacture of submicron metal barcodes for application as identification tags. These Nanobarcodes™ identification tags (NBCs), are prepared via the sequential electrodeposition of various metals within an alumina template. The striped particles grow as replicas of the pores of the membrane, and can be released from the membrane through chemical treatment. The striping pattern of NBCs can be read out via optical microscopy, and allows for the synthesis of large numbers of distinguishable tags. Herein, we address the synthesis of these NBCs and the use of software that can be used to identify specific NBCs in optical microscope images containing several distinct species of NBCs.

Encoded metal nanoparticle-based molecular beacons for multiplexed detection of DNA

In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing. We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence. Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid, unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched. By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays are possible using a single fluorophore, from a multiplexed RT-PCR reaction.

Use of Nanobarcodes® Particles in Bioassays

We have developed striped metal nanoparticles, Nanobarcodes particles, which can act as encoded substrates in multiplexed assays. These particles are metallic, encodeable, machine-readable, durable, submicron-sized tags. The power of this technology is that the particles are intrinsically encoded by virtue of the difference in reflectivity of adjacent metal stripes. This chapter describes protocols for the attachment of biological molecules, and the subsequent use of the Nanobarcodes particles in bioassays.

Microvolume Laser Scanning cytometry platform for biological marker discovery

Modern chemical synthesis and screening technologies have the ability to create large numbers of lead components but still do not answer questions of efficacy, dosing, toxicity and optimal patient population. SurroMed was founded to develop discovery technologies for new biological markers that will answer these questions. Biological markers will be derived from the results of many different assays; cell surface, serum factors and others, many performed using whole blood and other fluids and tissues. We report on the design of a Microvolume Laser Scanning Cytometer (MLSC) and disposable capillary arrays to be used in biological marker discovery. The MLSC machines are used primarily for cell surface assays, though they are suitable for other fluorescence assays as well. Each capillary requires a very small sample volume per assay, less than twenty micro- liters, and so allows hundreds of assays to be performed on a single ten milliliter blood draw. The new MLSC is capable of optimally detecting four fluorescence colors at different scan rates. HeNe excitation and red emission permits the use of whole blood, so that no lysing or cell separation is required. The MLSC instrument and disposable capillary arrays are in routine use for biological marker discovery at SurroMed.

Detection of biomolecules using nanoparticle surface enhanced Raman scattering tags

Detection tags based upon surface enhanced Raman scattering provide an alternative to the widely used fluorescence methods. Several aspects of these tags are presented in this report. The tags can be made to display many different spectra, thus they can be used for multiplexed detection schemes. They generate a large enough number of photons to be readily detected, and spectra acquired from mixtures of tags can be analyzed giving accurate amounts of the components. The surface of the tags can be easily modified to present common biological molecules (streptavidin and analogues). Finally, we demonstrate their use to quantitatively detect interleukin-4 (IL4) and interleukin-7 (IL7) in a microarray format.

Nanobarcodes particles as covert security tags for documents and product security

At Nanoplex Technologies, Inc. we have developed Nanobarcodes particles, which are encodeable, machine-readable, durable, sub-micron sized taggants which have application for document and product security. We will present results on the use of Nanobarcodes particles in a number of authentication and anti-counterfeiting applications. We also focus on the software component in recognition of particles imaged against varied backgrounds.