hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

George Mason University

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation.

Urine represents a valuable biofluid for noninvasive measurement of Human Growth Hormone (HGH) secretion. Unfortunately, currently available commercial HGH immunoassays do not achieve the sensitivity needed for urinary HGH measurement in the low picogram per milliliter range, the expected normal concentration range of HGH in urine. A nanotechnology based sample preprocessing step was used to extract and concentrate HGH in urine so that urinary HGH could be measured with a clinical grade standard immunoassay designed for serum (Immulite 1000, Siemens). We applied the nanoparticle enhanced immunoassay to evaluate the baseline value of urinary HGH in a population of healthy young adults (age 18-30, N=33, median 21, M: F=39%:61%, with no reported medical therapies). Nanoparticle sample preprocessing effectively improved the lower limit of detection of the Immulite HGH assay by more than 50 fold, shifting the linear range of the assay to encompass the expected value of urinary HGH. The full process between run and within run CV% was 7.9 and 9.0%, respectively. On 33 healthy volunteers, the 95% reference values for hGH in spot urine normalized to specific gravity were 0.64 - 16.85 pg/mL (0.05-5.82 ng/g creatinine). Nanoparticle preprocessing constitutes a reliable means of measuring urinary HGH with a clinical grade immunoassay, now establishing a normal baseline value for HGH in urine. Nanoparticles can be used to study the kinetics of HGH excretion in urine, and the factors that influence urinary HGH secretion and HGH isoform proportions.

Ruben Magni

Center for Applied Proteomics and Molecular Medicine

George Mason University

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Ruben Magni

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George Mason University

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Center for Applied Proteomics and Molecular Medicine