This showed that sample flow through the strip was complete. Open in a separate window Figure 5 Use of nanotags in a sandwich immunoassay for IgG. and multiplexing capabilities. Raman spectra can serve as a unique signal, or fingerprint, that can be leveraged for specific and sensitive detection of analytes. Even though Raman scattering is weak, the Raman signal of a molecule can be greatly enhanced by being in proximity to a roughened metal surface or a nanoparticle by several orders of magnitude, as high as 109.1?3 Consequently, SERS has become a powerful technique because of its high sensitivity to detect analytes,4 sometimes down to attomol levels. In particular, using SERS enhancement in the nanotag conformation has been useful for expanding the capabilities of biological sensing, imaging, and detection. Typically, a reporter molecule is conjugated to the surface of a nanoparticle,5?7 which provides the SERS signal. The nanoparticle is attached to a species that can bind to a biomolecule with specificity, such as an antibody, peptide, targeting ligand, or aptamer, thus enabling measurement of the presence of a biomolecule via the Raman signal of the reporter on the NP surface. This approach has been applied successfully for cell imaging,8?10 paper-based immunoassays,11?13 bead assays,14 and other biological applications.15,16 In addition, the ability to excite the Raman UDM-001651 reporters in the tissue window facilitates in vivo detection and imaging. 17 SERS becomes more powerful when it is highly multiplexed, UDM-001651 and thus approaches to expand the number of nanotags in an experiment have been pursued for techniques such as screening peptide libraries,14 sorting cell-binding species, multiplexed imaging, and many others.18,19 Fortunately, there is a multitude of Raman reporters that can be found in the literature, such as Raman dyes [e.g., malachite green (MG), methylene blue (MB), and crystal violet (CV)]20 which are widely used in cell imaging because of their intense signals. Thiolated molecules21 (e.g., 4-mercaptobenzoic acid (MBA), 4-methoxythiophenol) are commonly used because of their ability to conjugate directly to gold surfaces.22 In addition to these classes of molecules, there are a many other small molecules with characteristic Raman spectral features [e.g., 1,2-bis(4-pyridyl)ethylene (BPE)] that has made them well suited for SERS.23 Others have demonstrated multicolor SERS detection with combinations of a large number of reporters in bar coding approaches and have been able to successfully deconvolute the spectra of multiple reporters.24,25 Furthermore, strategies such as multiplexing with orthogonal measurement techniques such as fluorescence spectroscopy can introduce an even higher degree of diversity.16 However, the performance of a SERS multiplexed assay relies on the ability to deconvolute the signals from each of the reporters. Spectral overlap between reporter makes deconvolution more difficult, and thus reporters are chosen to have minimal overlap. Selecting these molecules is typically straightforward for situations which require only UDM-001651 one or two nanotags, as it is easy to find two reporters with minimally overlapping spectra, especially if they are small molecules.26 Unfortunately, achieving minimal spectral overlap becomes increasingly difficult when a large number of reporters are required. While this is straightforward for two reporters, this rapidly becomes more challenging UDM-001651 as the number of required reporters increases. This is further complicated by the use of larger molecules such as Raman dyes which have Mouse monoclonal to FOXA2 complicated spectra. The choice of Raman reporters can be a major limiting factor in multiplex design, and suboptimal reporter choice can compromise deconvolution and ultimately multiplexing capability. While multiplexed SERS has been achieved previously, a quantitative method for selecting a set of reporter molecules has not yet been detailed, and there is no generally accepted approach. Typically reporters are selected UDM-001651 based on the separation of their most prominent peaks, and are often eyeballed, which is not feasible for highly multiplexed assays. Thus, there is a need for a method for choosing and also evaluating an optimal set of reporters for their proper deconvolution. Furthermore, ratiometric information between analytes is often necessary for clinical assays, so the ability to quantify the contributions of the different reporter molecules is desirable.27 Here, we investigate a protocol for selecting a set of optimal reporters for a multiplexed SERS assay and their relative quantification that could serve as a method to provide differential diagnosis among diseases presenting distinct levels of the same biomarkers. This method is based on the use of a.
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