POS-383 PLASMONIC-FLUOR ENHANCEMENT OF KIDNEY INJURY BIOMARKER MICROARRAYS

Abstract

Biomolecular microarrays based on fluorescence read-out are important clinical and research tools, especially for simple, high-throughput and rapid proteomic analyses allowing miniaturization of tens to hundreds of assays on one small piece of analytical substrate. Despite advantages such as high multiplicity, rapid screening, and low sample volume, this methodology suffers from low sensitivity (even inferior to ELISA), which hinders its widespread application. Therefore, we investigated the applicability of plasmonic-fluors for enhancing the sensitivity of immunosandwich arrays A urine sample from a patient with chronic kidney disease was diluted using blocking/sample buffer, mixed with the biotinylated detection antibody cocktail, and added onto four nitrocellulose membrane arrays per the manufactures (Raybiotech) instructions for fluorescent detection using a LI-COR instrument with fluorophore streptavidin-800CW and then imaged/quantified on a LI-COR CLx (Figure left). Finally, plasmonic-fluor-800CW was added on the arrays and thoroughly rinsed to remove the unbound plasmonic nanoconstructs before reimaging. Also imaged is an array using R&D Systems human kidney array as analytical substrate and patient plasma as biospecimen source. Seven biomarkers were identifiable and quantifiable (albumin, β2-microglobulin, CCL-2/MCP-1, cystatin C, CXCL9, sTNFR1 and NGAL) (Spots D, G, L, M, P, S, T, respectively) using conventional fluor (Figure 1, left). Along with the three positive control Spots (A, B and W), the mean repeatability was 104% with a standard deviation of 4% for the four arrays Three of the arrays were then treated with plasmonic-fluor and reimaged (Figure 1, right). All 20 biomarkers (C-V) (KIM-1, albumin, osteopontin, trefoil factor-3, b2-microglobulin, clusterin, CXCL16, GPNMB, FABP-1, CCL-2/MCP-1, sTNFR1, calbindin-1, CXCL10, cystatin C, hepatocyte growth factor, CXCL9, NGAL, TIMP-1, and VCAM-1) were now quantifiable (Figure 1, right). Repeatability between the three arrays for all 46 spots either biomarker or control was a mean of 99% with a standard deviation of 5%. Comparing the relative densities of the biomarker and control spots found a 75-fold enhancement of the fluorescence by the plasmonic-fluor (red bars) over that of the standard fluor (blue bars) with a standard deviation of 8-fold. The reproducibility of the positive control spots (A, B and W) was 103% with a standard deviation of 5% without fluor and 6% with fluor. This demonstrates the reproducibility of the technique to identify biomarkers relevant to kidney injury and function and normalize biomarker levels from array to array. Similar results were obtained with kidney arrays from R&D Systems (Not Shown Here) using patient plasma. Additionally, since the plasmonic-fluor uses gold Nano rods, visible spots are quantifiable for abundant biomarkers while fluorescence-based analyses make the relative quantifiable dynamic range of all 38 biomarkers of interest of five-orders of magnitude. Use of plasmonic-fluor to enhance the sensitivity of multiplexed microarrays to detect biomarkers of interest allows relative quantification ofall analytes on the array, not just abundant markers. This technology can be adapted to analysis on smartphones extending analysis beyond central laboratories.