Non-invasive Raman spectroscopy of Albumin in the aqueous humor of the anterior chamber of the eye : an initial in vitro validation of a novel slit lamp mounted setup for Raman spectroscopy of proteins in the aqueous humor of the living eye using a customized eye model
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Introduction: Aqueous humor proteins have been identified as important biomarkers for ocular diseases. The main aim of this study was to perform an initial in vitro validation of a novel patented instrumental setup for non-invasive measurements of proteins in the aqueous humor of the anterior chamber of the human eye by means of an adapted Raman spectrometer mounted to a commercial slit lamp microscope. Methods: We used Bovine Serum Albumin (BSA) and a model eye for measurements with Raman spectroscopy: Twenty-five measurements related to the number of samples to obtain a noise cancelling effect on the Raman shift intensity, five related to repeatability, sixteen related to linearity and concentrations of Albumin within a physiologic range (i.e. 0.02 - 2.08 mg/ml). Spectral data preprocessing included a smoothing Savitsky-Golay of a polynomial order 2 and 3 smoothing points followed by a standard normal variate transformation for baseline correction and normalization. Principal component analysis with full cross validation and partial least square regression were run on the data to establish the predictive ability of the model. Results: Simulations showed that noise was sufficiently cancelled with twelve samples. The coefficient of variation was determined as 0.69 % indicating an excellent repeatability of the measurements in 1650 cm-1 band. Partial least square regression showed the ability of the patented instrumental setup to measure low concentrations of Albumin in the physiologic range down to 0.2 mg/ml with increasing certainty of linearity as the concentration increases. Conclusion: The results of the study have for the first time shown that a novel patented instrumental setup for Raman spectroscopy of the molecular content of the aqueous humor is able to provide measurements of Albumin at physiologic concentrations with a high degree of linearity and repeatability from a model eye.