Figure 1.
Plasmonic gold films for peptide microarrays.
A) Scanning electron microscopy (SEM) of an as-made plasmonic gold film containing nano-islands formed on a glass substrate. Inset: an X-ray photoelectron spectrum (XPS) of the film. B) SEM of a plasmonic gold film coated with a layer of avidin proteins (avidin coating caused blurred features of the underlying gold nano-islands). Inset, an XPS spectrum taken on the avidin coated gold substrate showing the presence of nitrogen (in the avidin) over the film. C) A schematic drawing of a multiplexed peptide array (two peptides are shown, red and blue; peptide spots are surrounded by PEG-star coating) on gold for antibody screening based on a 3-layer assay with a NIR fluorophore reporter. D) Intensity of IRDye800 fluorescence (emission ∼ 800 nm) measured on a layer of IRDye800 labeled avidin coated on glass vs. on a plamonic gold film. A ∼100 fold fluorescence enhancement was observed on gold.
Figure 2.
Multiplexed probing of peptide-antibody interaction on plasmonic gold vs.
glass. A) Schematic of a 4-plex histone peptide microarray containing unmodified and acetylated Histone H2B and H3 peptides immobilized on gold plasmonic and glass substrates for interacting with a mixture of 2 commercial antibodies against the acetylated H2B and acetylated H3 peptides in a solution (right photo). B) Top panel: fluorescence images of histone peptide microarrays on gold plasmonic substrate probed in titration with commercial antibodies targeting acetylated H2B and H3 peptide. Bottom Panel: identical peptide microarrays on streptavidin coated glass slides probed with the same commercial antibodies as top panel. C) and D) are calibration curves for antibody quantification generated by averaging the integrated fluorescence intensity of secondary antibody-IRDye800 emission over the 5 duplicate microarray spots for each of the histone peptide at various concentrations of histone antibodies on plasmonic gold slide and streptavidin-glass slide. Error bars represent the standard deviation of the mean over the 5 duplicate assay features.
Figure 3.
An integrated peptide-antigen microarray for profiling serum antibodies of 20 patients with systemic lupus erythematosus (SLE).
A) Peptide-antigen microarray layout including modified and unmodified histone H2A, H2B, H3, H4 peptides as well as whole antigens. Ac: acetylated; aa: amino acid; Me1: methylated; Me2: dimethylated; Me3: trimethylated; Ph: phosphorylated; K: Lysine; S: Serine. Number indicates amino acid position from the N-terminus of its corresponding histone proteins. Peptide sequences are listed in Table S1. B) Microarray imaging results of a SLE patient serum probed on a plasmonic gold substrate (left) vs. on a commercial streptavidin-glass substrate (right) shown in the same IRDye800 fluorescence intensity scale. C) Dynamic range of fluorescence signal reading among 20 SLE patients measured on each peptide and antigen spots on avidin/gold slide (upper panel) and commercial streptavidin/glass slide (lower panel). Each dot represents a fluorescence intensity measured on a peptide or antigen spot shown along the x-axis in the serum of each of the 20 SLE patients. A dot is drawn only when the fluorescence intensity is above the background by 2 standard deviation of the background signal.
Figure 4.
Peptide-antigen microarrays on plasmonic gold substrate for profiling antibodies in serum samples of SLE patients and healthy individuals.
A) Box plot of serum IgG antibody reactivity against several peptides and a whole antigen for 20 SLE patients and 20 healthy controls. Acetylated histone H2B peptides were found to be able to differentiate SLE patients and healthy controls (top box plots) together with whole H2B protein (bottom left box plot). While the H3 peptide with the 18th lysine methylated were found not capable of telling SLE patient from healthy control (bottom right box plot). B–D) Heatmaps displaying antibody reactivity to (B) histone peptides only, (C) whole antigens only, and (D) a combination of histone peptides and whole antigens that are identified capable of differentiating SLE patients and healthy controls with false discovery rate (q value) <0.001. The dashed lines are drawn to highlight the separation of SLE and healthy groups identified by using the average linkage Euclidean distance hierarchical clustering method. Color intensity of each grid in the heatmap reflected mean fluorescence intensity of corresponding peptide or antigen spot on the microarray for each SLE patient or healthy individual. In (B) and (C), several SLE patients (labeled in blue and purple color) are misplaced in the healthy group. These patients are grouped in the SLE side in (D) that profiles antibodies against both peptides and whole antigens. However, one healthy individual is mis-placed in the SLE group by this approach, reducing the specificity of this analysis.