SAT-144 AN ULTRASENSITIVE SURFACE ENHANCED RAMAN SCATTERING (SERS)-BASED ASSAY FOR KIDNEY INJURY MOLECULE-1 (KIM-1)

Abstract

Kidney injury molecule-1 (KIM-1) is a biomarker of acute and chronic kidney injury. However, dilution of urine samples to minimize the impact of pH and solute on results can push the concentration levels to below the lower limits detection of conventional ELISAs. Additionally, small samples from infants, children or from non-human renal injury models such as mice limits analyte detection. Here we develop an ultrasensitive (sub-pg/ml) assay utilizing surface enhanced Raman scattering (SERS) to measure KIM-1. Capture and detection antibodies of a commercially available KIM-1 ELISA kit (R&D Systems) were utilized. [email protected] nanocuboids ([email protected]) with 16 nm shell thickness, assembled on silicon wafers, were derivatized with cysteamine followed by glutaraldehyde to covalently attach the capture antibody. Separately, [email protected] were derivatized with the Raman reporter 4-aminothiophenol (pATP), glutaraldehyde followed by the detection antibody. After analyte capture on the silicon wafer, washing, incubating with the derivatized detection antibody and washing, the silicon wafers were subject to SERS. Among several prominent Raman bands, the intensity band at 1080 cm-1 of pATP was proportional to the concentration of human recombinant KIM-1 spiked into artificial urine. The urine of control individuals and patients with kidney disease was diluted prior to assay and the results compared to that using the conventional ELISA. The Raman intensity band at 1080 cm-1 of pATP was proportional to the KIM-1 concentration from to 400 pg/ml. As little as 1fg/ml above background could be reliably detected. The KIM-1 concentration, determined in patient urine using both the conventional ELISA and the SERS assay, was found to be synonymous with a correlation coefficient of 0.93 ( p<0.002). A similar strategy was used to develop a sub-pg/ml assay for the pro-inflammatory/myokine molecule IL-6. Using the power of SERS, capture and detection antibodies of a conventional ELISA kit were modified to measure sub-pg/ml amounts of KIM-1. This sensitivity allows dilution of samples to eliminate the influence of pH and/or solute concentration that would otherwise affect conventional ELISA results. Also the sensitivity of the assay allows the use of small samples of urine or plasma from infants, small children or from small animals such as mice with experimental renal diseases. It would be possible to detect preclinical changes in KIM-1 prior to disease manifestation. This assay strategy can be adapted to multiplexing to measure two or more analytes simultaneously in one sample by utilizing different Raman reporter molecules on different detection antibodies, an appropriate mix of capture antibodies on the silicon wafer, and monitoring non-overlapping Raman bands specific for each reporter.