Peptoid microsphere coatings to improve performance in sandwich ELISA microarrays

Enzyme-linked immunosorbent assay (ELISA) microarray performance is limited by low assay sensitivity and dynamic range. Increasing the surface area for reagent binding can help to improve performance, but standard techniques such as roughening the surface or adding a polymer coating lead to increased non-specific fluorescence and do not have reproducibly improved performance. Another approach to increase surface area is adding a microsphere coating on the surface. Poly-N-substituted glycine (peptoid) microspheres are ideal for this application due to low immunogenicity, protease-resistance, and biocompatibility. Peptoids are polymers with a backbone similar to peptides, but with the side chains appended to nitrogen rather than the alpha carbon. A variety of side chain chemistries can be incorporated into peptoids through a solid-phase, sequence-specific synthesis protocol. Here we report the development of sandwich ELISA microarray on peptoid microsphere coated glass slides. Coating morphology was evaluated via SEM and efficacy was assessed by ELISA microarray performance. Peptoid microsphere coated glass slides exhibit an increase in signal intensity and dynamic range as compared to commercially available microarray slides. These studies show the potential for peptoid microspheres as coatings for ELISA microarray slides, as well as for use in other biosensor applications

Uniform and Robust Peptoid Microsphere Coatings

Peptoids that are helical and partially water soluble have been shown to self-assemble into microspheres when the peptoid solution is dried on a silicon substrate. Such microsphere coatings have great potential for use in biosensor technologies, specifically to increase the surface area for binding. However, in order to be useful, the peptoids must consistently form uniform coatings. In this study we investigated the effects of various coating protocol parameters on the uniformity of the resulting peptoid microsphere coatings, including (i) solvent, (ii) administration technique, and (iii) drying environment. In addition, we investigated the robustness of the coatings as well as the potential for using a glass substrate. These studies show that uniform, robust peptoid microsphere coatings can be formed using protic solvents, a full coverage administration technique, and drying in open air on silicon or glass substrates

An Internal Calibration Method for Protein-Array Studies

Nuisance factors in a protein-array study add obfuscating variation to spot intensity measurements, diminishing the accuracy and precision of protein concentration predictions. The effects of nuisance factors may be reduced by design of experiments, and by estimating and then subtracting nuisance effects. Estimated nuisance effects also inform about the quality of the study and suggest refinements for future studies.We demonstrate a method to reduce nuisance effects by incorporating a non-interfering internal calibration in the study design and its complemental analysis of variance. We illustrate this method by applying a chip-level internal calibration in a biomarker discovery study.The variability of sample intensity estimates was reduced 16% to 92% with a median of 58%; confidence interval widths were reduced 8% to 70% with a median of 35%. Calibration diagnostics revealed processing nuisance trends potentially related to spot print order and chip location on a slide.The accuracy and precision of a protein-array study may be increased by incorporating a non-interfering internal calibration. Internal calibration modeling diagnostics improve confidence in study results and suggest process steps that may need refinement. Though developed for our protein-array studies, this internal calibration method is applicable to other targeted array-based studies.

Levels of plasma glycan-binding auto-IgG biomarkers improve the accuracy of prostate cancer diagnosis

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥ 2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment

Rationally Designed Peptoids Modulate Aggregation of Amyloid-Beta 40

Alzheimer’s disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States. Plaques composed of aggregated amyloid-beta protein (Aβ) accumulate between the neural cells in the brain and are associated with dementia and cellular death. Many strategies have been investigated to prevent Aβ self-assembly into disease-associated β-sheet amyloid aggregates; however, a promising therapeutic has not yet been identified. In this study, a peptoid-based mimic of the peptide KLVFF (residues 16–20 of Aβ) was tested for its ability to modulate Aβ aggregation. Peptoid JPT1 includes chiral, aromatic side chains to induce formation of a stable helical secondary structure that allows for greater interaction between the aromatic side chains and the cross β-sheet of Aβ. JPT1 was found to modulate Aβ40 aggregation, specifically decreasing lag time to β-sheet aggregate formation as well as the total number of fibrillar, β-sheet structured aggregates formed. These results suggest that peptoids may be able to limit the formation of Aβ aggregates that are associated with AD