A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs).

BACKGROUND: Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. METHODOLOGY/PRINCIPAL FINDINGS: To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. CONCLUSIONS/SIGNIFICANCE: Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

Raman nanoparticle probes for antibody-based protein detection in tissues.

Surface-enhanced Raman scattering (SERS) nanoparticles are emerging as a new approach for optical detection of biomolecules. In a model assay in formalin-fixed paraffin-embedded (FFPE) prostate tissue sections, we detect prostate-specific antigen (PSA) using antibody (Ab) conjugated to composite organic-inorganic nanoparticles (COINs), and we use identical staining protocols to compare COIN-Ab and Alexa-Ab conjugates in adjacent tissue sections. Spectral analysis illustrates the fundamental difference between fluorescence and Raman signatures and accurately extracts COIN probe signals from background autofluorescence. Probe signals are used to generate images of PSA expression on the tissue, and quality measures are presented to characterize the performance of the COIN assay in comparison to Alexa. Staining accuracy (ability to correctly identify PSA expression in epithelial cells) is somewhat less for COIN than Alexa, which is attributed to an elevated false negative rate of the COIN. However, COIN provided signal intensities comparable to Alexa, and good intra-, inter-, and lot-to-lot consistencies. Overall, COIN and Alexa detection reagents possess similar performance with FFPE tissues, supporting the further development of Raman probes for this application. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Spectral analysis of multiplex Raman probe signatures.

Raman nanoparticle probes are an emerging new class of optical labels for interrogation of physiological and pathological processes in bioassays, cells, and tissues. Although their unique emission signatures are ideal for multiplexing, the full potential of these probes has not been realized because conventional analysis methods are inadequate. We report a novel spectral fitting method that exploits the entire spectral signature to quantitatively extract individual probe signals from multiplex spectra. We evaluate the method in a series of multiplex assays using unconjugated and antibody-conjugated composite organic-inorganic nanoparticles (COINs). Results show sensitive multiplex detection of small signals (<2% of total signal) and similar detection limits in corresponding 4-plex and singlet plate binding assays. In a triplex assay on formalin-fixed human prostate tissue, two antibody-conjugated COINs and a conventional fluorophore are used to image expression of prostate-specific antigen, cytokeratin-18, and DNA. The spectral analysis method effectively removes tissue autofluorescence and other unknown background, allowing accurate and reproducible imaging (area under ROC curve 0.89 +/- 0.03) at subcellular spatial resolution. In all assay systems, the error attributable to spectral analysis constitutes

Composite organic-inorganic nanoparticles as Raman labels for tissue analysis.

Composite organic-inorganic nanoparticles (COINs) are novel optical labels for detection of biomolecules. We have previously developed methods to encapsulate COINs and to functionalize them with antibodies. Here we report the first steps toward application of COINs to the detection of proteins in human tissues. Two analytes, PSA and CK18, are detected simultaneously using two different COINs in a direct binding assay, and two different COINs are shown to simultaneously label PSA in tissue samples.