Surface-enhanced Raman scattering (SERS) is under active investigation in biomedical diagnostics due to its high sensitivity, increased levels of multiplexing, robustness, and ability to perform detection in blood and other biological matrices. SERS has been successfully applied for labeling cells 1 and tissues, 2 for multiplexed biomarker labeling to monitor apoptotic processes, 3 and for real-time monitoring of single live cell signaling processes. 4We have developed SERS tags (Nanoplex biotags, a trademark of Oxonica Inc.), comprised of one or more SERS-active metal nanoparticles (Au) and a sub-monolayer of reporter molecules adsorbed to the metal surface, all encapsulated in a protective and functionalized silica coat ( Figure 1A). 5 Nanoplex biotags have been successfully used in biological applications. 6,7 Here we describe a novel application of Nanoplex biotags for the direct detection of rare cancer cells in whole blood.Malignant cells are shed and circulate in the bloodstream of patients with solid tumors. 8 Since the "seed and soil" theory for circulating tumor cells (CTCs) was hypothesized 9 and confirmed, 10 two major approaches, based on polymerase chain reaction or cytometric methods (such as the CellSearch system), have been established for CTC detection. 8,11 However, high instrument cost and labor-intensive and time-consuming procedures remain a major concern and hamper their use in clinical diagnostics. Taking advantage of the intrinsic properties of the SERS tags, we have developed a novel, homogeneous, no-wash assay platform that overcomes the current assay limitations. We use magnetic beads for CTC capture and Nanoplex biotags for rapid and sensitive detection directly in human whole blood. Scheme 1 illustrates the concept in which magnetic beads, conjugated to an epithelial cell-specific antibody (epithelial cell adhesion molecule, anti-EpCAM), and the SERS tags, conjugated to an anti-her2 antibody (human epidermal growth factor receptor-2), bind to a tumor cell. Since the breast cancer cell is of epithelial origin, the magnetic bead-EpCAM antibody will specifically bind to this tumor cell but not regular circulating blood cells. Since the her2 receptor is highly expressed on the breast cancer cell membrane, the anti-her2-SERS tag will specifically recognize these tumor cells. By adding the magnetic bead-EpCAM and SERS tag-her2 conjugates to a patient's blood sample, circulating breast cancer cells (CTCs) can be detected rapidly and with good sensitivity in the presence of whole blood.In a proof-of-concept experiment, the breast cancer cell line SKBR3, expressing high levels of her2 receptor on the cell surface, 12 was used as a model target. After a 30 min incubation of SKBR3 cells with magnetic bead-EpCAM and SERS-her2 conjugates, small volumes of the reaction mixtures were loaded on a glass slide. Bright-field microscopy imaging showed specific binding of the magnetic beads-EpCAM to the tumor cells ( Figure 1B), and anti-her2

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable nowash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase ΔG of folding in 500 mM buffered NaCl of approximately −4 kcal/mol performed better than those with ΔG > −2 or < −6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 °C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with K d = 1.7 ± 0.3 nM. The detection limit was ∼100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

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.(Nanobiotechnology

The rapid growth and development in biodetection technology has largely been driven by the emergence of new and deadly infectious diseases and the realization of biological warfare as new means of terrorism. [1,2] To address the need for portable, multiplex biodetection systems, we report here a novel biosensing platform using engineered nanowires as an alternative substrate for sandwich immunoassays (Figure 1 A). The nanowires are built through submicrometer layering of different metals by electrodeposition within a porous alumina template. [3,4] A variety of metals can be deposited: in this study, we employed stripes of gold, silver, and nickel. Owing to the permutations in which the metals can be deposited, a large number of unique yet easily identifiable encoded nanowires can be included in a multiplex array format.Image processing of an optical reflectance image can enable the stripe pattern to be identified rapidly, while fluorescence images report information on the degree of binding between the antibody-conjugated nanowires and a fluorophore-tagged antigen target. Such nanowires have been utilized to efficiently detect and report both DNA hybridization and immunoassay processes. [5,6] Herein, we demonstrate the feasibility of using multistriped metallic nanowires (Figure 1 A) in a suspended format to enable rapid and sensitive single and multiplex immunoassays for biowarfare agent simulants.Both the hybridization and kinetics of the capture of the target analyte in solution favor the nanowires over conventional fixed array-based formats. The incorporation of an appropriate ferromagnetic metallic component, for example, Ni, enables the nanoparticles to be manipulated by using magnetic fields. [7][8][9][10] To demonstrate the capability of directly detecting potential biological warfare agents in both clinical and environmental samples, a reagent set of three antigens generally accepted for use in simulating actual biothreat agents was chosen. The three nonpathogenic simulants include 1) Bacillus globigii (Bg) spores to simulate Bacillus anthracis and other bacterial species, 2) RNA MS2 bacteriophage to simulate Variola (virus for smallpox) and other pathogenic viruses, and 3) ovalbumin (Ova) protein to simulate protein toxins such as ricin or botulinum toxin. Besides the relative handling safety of these simulants, they were also chosen to reflect the variation in target sizes, ranging from large bacterial spores ( % 2 mm) to small protein molecules ( % 2 nm). Figure 1. A) Analogy between a conventional barcode and a metallic stripeencoded nanowire (diameter % 250 nm; length % 6 mm). Ni segments (50 nm) are deposited at both ends on the magnetic nanowire (not drawn to scale). B) Schematic of the sandwich immunoassay performed on a nanowire. C) Post-assay reflectance and fluorescence readout of the nanowires. The identity of the antigen present can be easily identified from the stripe pattern of the nanowires; for example, the fluorescently lit nanowire to which anti-Bg spore Ab was attached has a stripe pa...