Prakasa Rao Aruna

Steady-state and time-resolved fluorescence spectroscopic characterization of urine of healthy subjects and cervical cancer patients

Abstract. Steady-state and time-resolved fluorescence spectroscopy were employed in the discrimination of cervical cancer patients from healthy subjects using urine samples. Fluorescence emission at 390 and 440 nm was considered to monitor the fluorescence of indoxyl sulfate and neopterin. Significant spectral differences were observed between healthy and cancer subjects. Different ratio parameters were calculated from the spectral intensity at 280- and 350-nm excitation and were subjected to stepwise linear discriminant analysis. In total, 84.0% of samples were correctly classified at 280 nm and 96.4% were correctly classified at 350 nm. The fluorescence decay kinetics of urine samples at 390-nm emission was best described by bi- exponential fits, whereas the decay characteristics at 440 nm of urine samples was best explained by bi-exponential fits and, in some cases, by tri-exponential fits. However, the decay kinetics of both indoxyl sulfate and neopterin standards was well described by bi-exponential decays. Based on the fluorescence emission characteristics and statistical analysis, the fluorophores indoxyl sulfate, neopterin, and riboflavin may be considered as potential biomarkers for cervical cancer diagnosis.

In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

Characterization of cervical normal and abnormal tissues by synchronous luminescence spectroscopy

Fluorescence spectroscopy has emerged as a promising modality in the discrimination of normal from pathologically diseased cells and tissues. As tissues are highly heterogeneous with many native fluorophores, emission spectra at one or more excitation wavelengths or excitation spectra corresponding to one or more emission wavelengths are used for diagnostic purpose. This could be overcome to great extent by applying synchronous luminescence spectroscopy. In the present study, synchronous luminescence fluorescence spectra of normal, pre malignant and malignant cervical tissues are measured by scanning both excitation and emission monochromator simultaneously with a wavelength difference of 20nm. The synchronous luminescence spectra of normal, pre-malignant and malignant cervical tissues shows the distinct peaks around 300, 350, 525nm with broad peak around 460nm and this may be due to tryptophan, collagen and flavin respectively. The broad band around 460nm may be due to the presence of pyridoxal phosphate, carotenes and lipopigments. Spectral data are also evaluated by both empirical and statistical analysis. Among the various analysis partial least square analysis provides better accuracy than other analysis in the discrimination of normal from abnormal tissues.

Ultraviolet fluorescence spectroscopy of blood plasma in the discrimination of cancer from normal

Native fluorescence spectroscopy of biomolecules has emerged as an intrinsic parameter in the characterization of the physiological state and the discrimination of pathological from normal conditions of cells and tissues. The key fluorescing biomolecules inc ells and tissues ar tryptophan, tyrosine, phenylalanine, collagen, elastin, NADH, flavin and porphyrin. Extensive studies were made on tissues of various origin to discriminate the malignancy from normal. The differences in the fluorescence emission spectra have been shown to separate benign and malignant tissues. In the present work, a pilot study was carried out on the characterization of blood plasma of both normal and cancerous subjects. The blood plasma was separated by centrifuging the blood and it was diluted in PBS by adjusting the O.D. to 0.5 at 280 nm. This diluted sample as excited in the UV region between 250-340 nm. Among the various excitation wavelengths, emission spectrum at 300 nm excitation has considerable difference between blood plasma of normal subjects and cancer patients. To quantify these differences and to verify if there is any diagnostic potential exists, the ratio of fluorescence intensities at 340 and 440 nm was calculated. It is found that the ratio value of normal blood plasma is less than 11 and for tumor, it is greater than 11. Besides, it is found that the ratio value of blood plasma from patients with cancer varies from 11 to 28, depending upon the stage of malignancy.

Raman spectroscopic characterization of blood plasma of oral cancer

Raman Spectroscopy is a versatile technique to probe in to the vibrational or rotational transitions of a molecule and extract complete information about the biochemical composition of the sample under investigation. The metabolic end products of the cell that were released in to the circulating blood would change the biological molecules and thus alter their spectral signatures. Based on this, a pilot study was carried out to discriminate oral cancer patients (n=14) from that of healthy subjects (n=14), using the Raman spectroscopy of blood plasma using Lab RAM HR 800 (HORIBA Jobin Yvon, France) instrument for the spectral range 800- 1800 cm-1. From the spectral profiles of the bio constituents such as protein, amino acids, glucose and lipid, markable difference was observed between the cancer patients and the healthy subjects. Raman peaks corresponding to bio components such as phenylalanine in albumin (939, 1002 cm-1), glucose (1124 cm-1), tryptophan (1205 & 1342 cm-1), amide III (1247 cm-1) and phospholipids (1450 & 1656 cm-1) were different for oral cancer patients from that of healthy subjects. The averaged spectra of cancer patients showed a noticeable blue shift for the peaks corresponding to protein at 1002 cm-1, lipid at 1450 and 1656 cm-1 and a overall decrease in intensity was observed for cancer spectrum. Further, the Raman spectra were analyzed employing the multivariant statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA), for the range 800- 1800 cm-1. The results were promising in discriminating blood plasma of oral cancer patients from that of healthy ones.

Influence of Anionic Surface Charged Biocompatible Dendrimer With a Photosensitizer, Protoporphyrin IX, on Human Red Blood Cells: A Spectroscopic Investigation

A biocompatible anionic dendrimer with carboxylic acid and phenolic hydroxyl functional groups at the surface of every half and full generations was designed by condensing phloroglucinol and succinic acid and its hemolytic effect on red blood cells was studied. The study reveals that the anionic surface charged dendrimer exhibits hemocompatibility and satisfies the reduction of dark toxicity of photosensitizer when loaded inside the nanocarrier, one of the prime requirements for a drug delivery system in photodynamic therapy applications. GRAPHICAL ABSTRACT

Steady state and time-resolved fluorescence spectroscopic characterization of normal and cancerous urine

Urine is one of the diagnostically important bio fluids, as it has many metabolites and some of them are native fluorophores. There may be a variation in the distribution and the physiochemical properties of the fluorophores during any metabolic change and pathologic conditions. Native fluorescence spectroscopy has been considered as a promising tool to characterize the fluorophores present in the urine. In this study, we aimed at characterizing the urine of both normal and patients with confirmed cancer using steady state and time-resolved fluorescence spectroscopy at 280 nm and 350 nm excitation. It is observed that the metabolites indoxyl sulphate and neopterin and its derivatives are responsible for altered spectral signatures at 280 nm, and 350 nm excitation. The overall spectral data were subjected to Principal Component Analysis and the resultant components were used as input in the linear discriminant analysis. As a total, 84% and 81.8% of samples were correctly classified at 280 nm and 350 nm respectively.

Native fluorescence spectroscopy of cervical tissues: classification by different statistical methods

Optical Spectroscopy in the diagnosis of diseases has attracted the medical community due to their minimally invasive nature. Among various optical spectroscopic techniques, native fluorescence spectroscopy has emerged as a potential tool in diagnostic oncology. However, still the reasons for the altered spectral signatures between normal and cancer tissues not yet completely understood. Recently, data reported that emission due to the alteration of some proteins is responsible for the transformation of normal in to malignant one. In this regard, the present study is aimed to characterize the native fluorescence spectroscopy of abnormal and normal cervical tissues, at 280nm excitation. From the study, it is observed that the normal and pathologically diseased cervical tissues have their peak emission around 339 and 336nm respectively with a secondary peak around 440nm. The FWHM value of emission spectra of abnormal tissues is lower than that of normal tissues. The fluorescence spectra of normal and various pathological conditions of cancerous tissues were analyzed by various empirical and statistical methods. Among various type of discriminant analysis, combination of ratio values and linear discrimination method provides better discrimination of normal from pre-malignant and malignant tissues.

Steady state fluorescence spectroscopic characterization of normal and diabetic urine at selective excitation wavelength 280 nm

Urine is considered diagnostically important for tits native fluorophores and they vary in their distribution, concentration and physiochemical properties, depending upon the metabolic condition of the subject. In this study, we have made an attempt, to characterize the urine of normal subject and diabetic patients under medication by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed employing the multivariate statistical method linear discriminant analysis (LDA) using leave one out cross validation method. The results were promising in discriminating diabetic urine from that of normal urine. This study in future may be extended to check the feasibility in ruling out the coexisting disorders such as cancer.

Photodynamic therapy of oral leukoplakia and oral lichen planus using methylene blue: A pilot study

Premalignant lesions like oral lichen planus (OLP), oral leukoplakia (OL) has a fair probability of transforming into malignancy and they are perverse toward conventional therapies. Photodynamic therapy (PDT) has been considered as an alternative/complimentary therapeutic modality for the management of premalignant lesions. In this study, methylene blue-mediated photodynamic therapy (MB-PDT) was used as a possible alternative method for the treatment of OLP and OL. A total of 15 OLP lesions and 13 OL lesions were enrolled in the study. The patients were irradiated using metal halide lamp filtered at 630 ± 10 nm, with a light exposure dose of 120 J/cm2 per sitting. For the OLP lesions, MB-PDT was performed once a week for four weeks and for the OL lesions, MB-PDT was performed twice a week for three weeks. Lesions were evaluated pre- and post- and at follow-up sessions by changes in sign and symptom scores, and size of lesions. We have observed a 53.3% of complete reduction in the treated OLP lesions and their decrease in size, sign and symptom score after treatment and at follow-up session was statistically significant. We have also observed complete response for one OL lesion of the 13 treated lesions. The result indicates that MB-PDT is an effective modality in management of OLP and OL. Among the two types of premalignancies treated with MB-PDT, OLP lesions responded much better than that of OL.