Sensitivity enhancement of a grating-based surface plasmon-coupled emission (SPCE) bionsor chip using gold thickness.

We describe a novel approach to enhance the sensitivity of a grating-based surface plasmon-coupled emission (SPCE) sensor by increasing the thickness of the metal film used in this system. The calculated optical properties of grating-based SPR spectra were significantly affected by both grating depth and by gold thickness. Higher angular sensitivity could be achieved at short wavelengths and under in situ measurement (analysis under aqueous condition). We confirmed the predicated enhancements of SPCE response using Alexa Fluor 647-labeled anti-mouse IgG immobilized on the SPCE sensor chips. Grating-coupled SPCE sensor chips can be used as a useful tool for high contents analysis of chemical and biomolecular interactions.

Highly sensitive grating coupler-based surface plasmon-coupled emission (SPCE) biosensor for immunoassay.

We have evaluated the performance of a novel high-content gold grating coupler-based surface plasmon-coupled emission (SPCE) biosensor system. The polyelectrolyte (PEL) layer on the system's biosensor chip surface was formed on the gold surface using a layer-by-layer (LbL) deposition technique to spatially separate fluorophores from the gold surface and as a linker layer for protein immobilization. The characteristics of the PEL spacer layers were determined by surface plasmon resonance (SPR) analysis and by ellipsometry. The SPCE response decreased as the spacer layer thickness increased above a dominant quenching range (10 nm). Two PEL layers of poly(diallyldimethylammonium chloride) and poly(acrylic acid) were found to be an effective spacer that minimized the quenching effect while maximizing SPCE responses for both direct and sandwich immunoassay formats. A mouse IgG sandwich immunoassay using this optimized spacer layer configuration showed a dose-dependent response with a 1 pg ml(-1) limit of detection by the 3σ rule. A nine log dynamic range of human IgG concentrations in unfractionated human serum could be analyzed using this approach. These results show the potential of the grating coupler-based SPCE biosensor as a sensitive platform for high-content analysis.

Analysis of immunoarrays using a gold grating-based dual mode surface plasmon-coupled emission (SPCE) sensor chip.

We have developed a novel dual mode immunoassay platform that combines the advantages of real-time, label free measurement of surface plasmon resonance (SPR) and the highly directional surface plasmon-coupled emission (SPCE) using a gold grating-based sensor chip. Since only fluorophore-labeled analyte molecules that are close to the metal surface of the sensor chip will couple to the surface plasmon, SPCE detection is highly surface-specific leading to background suppression and increased sensitivity. Theoretical calculations were done to find SPR and SPCE angles for a sensor chip optimized for Alexa Fluor 647. We have confirmed the SPR and SPCE responses on the dual mode sensor chip using Alexa Fluor 647 labeled anti-mouse IgG. Signal fluctuation of the dual mode sensor chip reader was below 1.2% and 0.8% for SPR and SPCE, respectively. The SPR response in this configuration showed a minimum detection level of 1 µg ml(-1), and the SPCE response showed a minimum detection level of 1 ng ml(-1) for the same sample. A range of human IgG concentrations in human serum was also analyzed with the dual mode sensor chip. The SPCE measurement is more sensitive than the SPR real-time measurement, and substantially extends the dynamic range of the assay platform, as well as enabling independent measurements of co-localized analytes on the same sensor chip region of interest. Since this assay platform is capable of measuring more than 1000 spatially encoded regions of interest on a 1 cm(2) sensor chip, it has the potential for high-content analyses of biological samples with both research and clinical applications.

Antigen-specific T cell phenotyping microarrays using grating coupled surface plasmon resonance imaging and surface plasmon coupled emission.

The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities were used to detect and characterize CD4(+) T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabeled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.