The broader impact of this Small Business Innovation Research (SBIR) Phase I project is the determination of the most accurate method of measuring protein abundance in patient samples. The answers obtained will address a fatal neonatal gut disease, necrotizing enterocolitis, that disproportionately affects African-American preterm infants and lacks disease-modifying treatments. The proposed technology will serve as a clinically-deployable diagnostic for hospitals, reference labs, and drug companies, particularly high-acuity neonatal intensive care units. The proposed project will advance the development of a diagnostic for an underserved population. In addition, the development team will include underrepresented innovators. The proposed project will optimize the choice of reference standard and detection method for protein abundance. Absolute quantification is a prerequisite for data interpretation and validation between experiments, laboratories, and testing platforms. Current clinical practice exploits only a single type of mutation that gives rise to disease; rarely do they address a target protein with extensive polymorphic variation that is age- and race-dependent. The goal of this proposal is to develop reference clinically robust standards to enable use of a new candidate biomarker in hospital pathology settings. Research objectives include: (1) identification of optimal reference standard composition for two common methods to quantify biomolecules in clinical settings and (2) understanding usage limitations of these reference standards in the background of high sequence variation in the human population.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.