Quantification and Identification of Microproteinuria using Ultrafiltration and ATR-FTIR Spectroscopy.

The presence of low amounts of specific proteins in urine can be an indicator of diagnosis and prognosis of several diseases including renal failure and cancer. Hence, there is an urgent need for Point-of-Care (PoC) methods, which can quantify microproteinuria levels (30-300 ppm) and identify the major proteins associated with the microproteinuria. In this study, we coupled ultracentrifugation with Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) to identify and quantify proteins in urine at low ppm levels. The process involves the pre-concentration of proteins from 500 µL of urine using an ultrafiltration device. After several washings, the isolated proteins are dried onto the ATR crystal forming a thin film. Imaging studies showed that the absorbance of the protein bands was linear with the amount of mass deposited on the crystal. The methodology was first evaluated with artificial urine spiked with 30-300 ppm of albumin. The calibration showed acceptable linearity (R2=0.97) and a limit of detection of 6.7 ppm. Linear relationships were also observed from urine of healthy subjects spiked with microproteinuria concentrations of albumin, immunoglobulin and haemoglobin giving a prediction error of the spiked concentration of 23 ppm. When multiple proteins were spiked into the real urine, multivariate analysis was able to decompose the dataset into the different proteins, but the multicomponent evaluation was challenging for proteins at low levels. Although the introduction of a pre-processing step reduces the PoC capability of the method, it largely increases its performance, showing great potential as a tool for the diagnosis and prognosis of several illnesses affecting urine proteic composition.