P0666BRILLOUIN AND RAMAN SPECTROSCOPIES FOR NON-CONTACT ASSESSMENT OF MECHANO-CHEMICAL PROPORTIES OF URINARY PROTEINS: A PROOF OF CONCEPT STUDY

Abstract

Abstract Background and Aims Proteinuria is a major marker of chronic kidney disease (CKD) severity, the predictor of renal function decline and cardiovascular mortality. The main pathway of CKD progressive is caused by tubular toxicity of filtering proteins, so more, the rapid development of renal failure is expected in those patients who have higher level of proteinuria. However, despite high level of proteinuria, paradoxically, some patients may have slow decline of renal function compared to those patients with lower level of urinary protein excretion. This may possibly due to their different mechanical (visco-elastic) and chemical properties, as well as the proteomics of excreting urinary proteins. Brillouin light scattering (BLS) and surface enhanced Raman scattering (SERS) spectroscopies are non-contact (laser optical-based) techniques providing visco-elastic and chemical property information of probed human biofluids. We aimed to study and compare the visco-elastic and chemical properties of urinary proteins using BLS and SERS spectroscopies in nephrotic patient with high protein excretion and healthy subject. Method Two urine samples from nephrotic patient (proteinuria>3.5 g/24h) and healthy subject (undetectable proteinuria) investigated using BLS and SERS spectroscopies. Brillouin spectroscopy performed using Torus single longitudinal mode laser in conjunction with high contrast JRS Instruments 6-pass tandem Fabry-Perot interferometer coupled with confocal microscope. Using Horiba LabRAM HR Raman microscope with 785 nm laser excitation andx100 microscope objective the SERS spectroscopy was performed on urine samples in contact 60 nm gold nanoparticles placed on gold film. Additionally, reversed-phase liquid chromatography-mass spectrometry (LC-MS) was conducted for profiling of urinary proteins LC-MS data was analysed by Mascot software using the SwissProt protein database. Results We found that elevated protein levels in urine alter the urine fluid elasticity sufficiently to be measurable by BLS spectroscopy, with urine from healthy and proteinuric subjects distinguishable to marked significance, which increases with sample concentration. BLS spectroscopy demonstrated that patients with High Protein Loss, characterized by stiffer urine (i.e. higher Brillouin shift and elastic modulus) and less viscous (i.e. less hypersound attenuation) compared to healthy subject. (Figure 1A). SERS measurements of two urine samples taken from patient with high protein loss in healthy subject (Figure 1B) showed the integrated Raman intensity ratio of 1338 cm-1 and 1002 cm-1 peaks is many times higher for the sample with high protein concentration vs sample with low protein concentration. Urinary protein sequencing and Mascot analysis (Table 1) demonstrated that proteinuric patient had different type of proteins with up to 187k Daltons of molecular weight. Conclusion In this proof of concept study, we demonstrated that laser-optical BLS and SERS techniques combined with LC-MS measurements revealed that human urine fluids of healthy and proteinuric subjects had strong variation in the visco-elastic and chemical properties coupled with various level of protein content.