The design of a portable Raman/SERS‐LFA reader with line illumination using a custom‐made fiber optic probe for rapid, quantitative, and ultrasensitive point‐of‐care testing (POCT) is presented. The pregnancy hormone human chorionic gonadotropin (hCG) is detectable in clinical samples within only 2–5 s down to approximately 1.6 mIU mL
−1
. This acquisition time is several orders of magnitude shorter than those of existing approaches requiring expensive Raman instrumentation, and the method is 15‐times more sensitive than a commercially available lateral flow assay (LFA) as the gold standard. The SERS‐LFA technology paves the way for affordable, quantitative, and ultrasensitive POCT with multiplexing potential in real‐world applications, ranging from clinical chemistry to food and environmental analysis as well as drug and biowarfare agent testing.
A biocompatible, seed-mediated synthesis of monodisperse ~60 nm gold nanostars, followed by hydrophilic stabilization with ethylene glycol-modified Raman reporter molecules, is presented. Their application as SERS labels for imaging of the tumor suppressor p63 in prostate biopsies by immuno-SERS microscopy is demonstrated.
The synthesis of 3D self-assembled plasmonic superstructures of gold nanospheres as well as the characterization of their structural and optical properties at the single-particle level is presented. This experimental work is complemented by FEM (finite element method) simulations of elastic scattering spectra and the spatial |E|(4) distribution for establishing structure-activity correlations in these complex 3D nanoclusters.
Activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory, and cytoprotective properties. Using recombinant APC, we identified a class of single-stranded DNA aptamers (HS02) that selectively bind to APC with high affinity. Interaction of HS02 with APC modulates the protease activity in a way such that the anticoagulant functions of APC are inhibited and its reactivity toward the protein C inhibitor is augmented in a glysoaminoglycan-like fashion, whereas APC's antiapoptotic and cytoprotective functions remain unaffected. Based on these data, the binding site of HS02 was localized to the basic exosite of APC. These characteristics render the exosite-specific aptamers a promising tool for the development of APC assays and a potential therapeutic agent applicable for the selective control of APC's anticoagulant activity.
SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.
Rationally designed gold/silver nanoshells (Au/Ag-NS) with plasmon resonances optimized for red laser excitation in order to minimize autofluorescence from clinical samples exhibit scattering cross-sections, which are ca. one order of magnitude larger compared with solid quasi-spherical gold nanoparticles (Au-NPs) of the same size. Hydrophilic stabilization and sterical accessibility for subsequent bioconjugation of Au/Ag-NS is achieved by coating their surface with a self-assembled monolayer (SAM) of rationally designed Raman reporter molecules comprising terminal mono-and tri-ethylene glycol (EG) spacers, respectively. The stability of the hydrophilically stabilized metal colloid was tested under different conditions. In contrast to metal colloids coated with a SAM without terminal EG spacers, the hydrophilically stabilized SERS particles do not aggregate under physiologically relevant conditions, i.e., buffer solutions with high ionic strength. Using these rationally designed SERS particles in conjunction with a microspectroscopic acquisition scheme, a sandwich immunoassay for the sensitive detection of interleukin-6 (IL-6) was developed. Several control experiments demonstrate the high specificity of the assay towards IL-6, with a lowest detectable concentration of ca. 1 pg mL À1 . The signal strength of the Au/Ag-NS is at least one order of magnitude higher compared with hydrophilically stabilized, non-aggregated solid quasi-spherical Au-NPs of the same size.
Rapid parallel detection of two cytokines (IL-6 and IL-8) with femtogram sensitivity in a simple direct dot-blot assay is demonstrated. The microspectroscopic SERS acquisition scheme employs rationally designed, hydrophilically stabilized Au-Ag nanoshells as SERS labels, which are optimized for signal enhancement upon red laser excitation.
Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of target proteins in tissue specimens by conventional IHC is achieved with dye- or enzyme-labeled antibodies in combination with light microscopy. In this contribution, we demonstrate the proof-of-principle for IHC based on surface-enhanced coherent Raman scattering for contrast generation. Specifically, antibody-labeled metallic nanoshells in conjunction with surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy are employed for the selective, sensitive, and rapid localization of the basal cell protein p63 in normal prostate tissue. Negative control experiments were performed in order to confirm the selective binding of the target-specific metal nanoprobes and to disentangle the role of plasmonic (metal) and molecular (Raman reporter) resonances in this plasmon-assisted four-wave mixing technique.
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