Non-invasive ocular biomarkers for early diagnosis of diseases
Materials and methods are disclosed for screening advanced glycation end-products from mammalian ocular lens proteins to quantify early biomarkers for the diagnosis of diseases such as diabetes mellitus and to prevent related complications.
1. A method for detecting and characterizing fluorophores in an ocular protein, cell or tissue, the method comprising:
(a) focusing an excitation light beam on the ocular protein, cell or tissue;
(b) detecting a fluorescent light emitted from the ocular protein, cell or tissue in a one or more wavebands, by an intensity of fluorescence from the ocular protein, cell or tissue;
(c) calculating the intensity of fluorescence for each waveband, wherein the calculation detects the fluorophore in the ocular protein, cell or tissue;
(d) measuring fluorescence decay with time after a short excitation pulse from the ocular protein, cell or tissue in the specific waveband; and
(e) calculating the fluorescence lifetimes from the fluorescent decay curves using global 1-4 three exponential fitting analysis.
2. The method of claim 1 , wherein the fitting analysis is calculated based on good autocorrelation function around zero, weighted number of residuals randomly distributed between +3 and −3, reduced chi square values between 0.9-1.1 and Durbin-Watson parameters of greater than or equal to 1.6, 1.7 and 1.8 for one, two and three exponential decays respectively.
3. An in vitro diagnostic test using the method of claim 1 for distinguishing the normal samples from pathological samples, further defined as based on the fluorescence intensity, decay and change in lifetime measurements at specific excitation wavelengths between 280-600 nm, and the damage to the proteins in the tissue is confirmed based on mass spectrometry and X-ray scattering.
4. The method of claim 1 , wherein the fluorophores are selected from the group consisting of A2E, lipofuscin, FAD, NADH, photo-degradation products of tryptophan and advanced glycation end-products.
5. The method of claim 1 , wherein results of calculating the intensity of fluorescence for each waveband distinguished normal from pathological cells or tissues based on differences in the fluorescence intensity maps.
6. The method of claim 1 , wherein detecting comprises screening changes in fluorophores over time and in response to treatments for a condition or disease.
7. The method of claim 6 , wherein the disease is selected from the group consisting of diabetes mellitus and diabetic retinopathy.
8. The method of claim 4 , wherein the photo-degradation products of tryptophan are selected from the group consisting of N-formyl kynurenine, anthranilic acid, kynurenine, 3-hydroxykynurenine and harmane.