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Zamudio, Spain

Laguna M.F.,Technical University of Madrid | Holgado M.,Technical University of Madrid | Santamaria B.,Technical University of Madrid | Lopez A.,Technical University of Madrid | And 11 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2015

Biophotonic Sensing Cells (BICELLs) are demonstrated to be an efficient technology for label-free biosensing and in concrete for evaluating dry eye diseases. The main advantage of BICELLs is its capability to be used by dropping directly a tear into the sensing surface without the need of complex microfluidics systems. Among this advantage, compact Point of Care read-out device is employed with the capability of evaluating different types of BICELLs packaged on Biochip-Kits that can be fabricated by using different sensing surfaces material. In this paper, we evaluate the performance of the combination of three sensing surface materials: (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), SU-8 resist and Nitrocellulose (NC) for two different biomarkers relevant for dry eye diseases: PRDX-5 and ANXA-11. © 2015 SPIE. Source

Laguna M.F.,Technical University of Madrid | Sanza F.J.,Technical University of Madrid | Soria J.,Bioftalmik | Jara M.,Antibody Bcn. Edifici Eureka | And 5 more authors.
Sensors and Actuators, B: Chemical | Year: 2014

The use of Biophotonic Sensing Cells (BICELLs) based on micro-patterned photonic architectures has been recently proven as an efficient methodology for label-free biosensing by using optical interrogation. According to this, different optical responses for a specific typology of BICELL, consisting of structures of SU-8 (pillars) were studied. This material is biocompatible with different types of biomolecules, which can be immobilized on its sensing surface as biomarkers. Different proteins were validated as biomarkers involved with dry eye dysfunction: PRDX5, ANXA1, ANXA11, CST4, PLAA y S100A6. For the first time, one of these biomarkers (PLAA) was immobilized and recognized by means of BICELLs as proof of concept. With the help of a simplified 1-D theoretical model recently published, it was estimated the thickness of the PLAA biofilm immobilized onto the sensing surface of BICELLs. © 2014 Elsevier B.V. Source

Gonzalez N.,Bioftalmik | Iloro I.,CIC Biomagune | Duran J.A.,Instituto Clinico Quirurgico Of Oftalmologia Icqo | Duran J.A.,University of the Basque Country | And 2 more authors.
Molecular Vision | Year: 2012

Purpose: To characterize the tear film peptidome and low molecular weight protein profiles of healthy control individuals, and to evaluate changes due to day-to-day and individual variation and tear collection methods, by using solid phase extraction coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling. Methods: The tear protein profiles of six healthy volunteers were analyzed over seven days and inter-day and interindividual variability was evaluated. The bilaterality of tear film and the effect of tear collection methods on protein profiles were also analyzed in some of these patients. MALDI-TOF MS analyses were performed on tear samples purified by using a solid phase extraction (SPE) method based on C18 functionalized magnetic beads for peptide and low molecular weight protein enrichment, focusing spectra acquisition on the 1 to 20 kDa range. Spectra were analyzed using principal component analysis (PCA) with MultiExperiment Viewer (TMeV) software. Volunteers were examined in terms of tear production status (Schirmer I test), clinical assessment of palpebral lids and meibomian glands, and a subjective OSD questionnaire before tear collection by a glass micro-capillary. Results: Analysis of peptides and proteins in the 1-20 kDa range showed no significant inter-day differences in tear samples collected from six healthy individuals during seven days of monitoring, but revealed subtle intrinsic interindividual differences. Profile analyses of tears collected from the right and left eyes confirmed tear bilaterality in four healthy patients. The addition of physiologic serum for tear sample collection did not affect the peptide and small protein profiles with respect to the number of resolved peaks, but it did reduce the signal intensity of the peaks, and increased variability. Magnetic beads were found to be a suitable method for tear film purification for the profiling study. Conclusions: No significant variability in tear peptide and protein profiles below 20 kDa was found in healthy controls over a seven day period, nor in right versus left eye profiles from the same individual. Subtle inter-individual differences can be observed upon tear profiling analysis and confirm intrinsic variability between control subjects. Addition of physiologic serum for tear collection affects the proteome and peptidome in terms of peak intensities, but not in the composition of the profiles themselves. This work shows that MALDI-TOF MS coupled with C18 magnetic beads is an effective and reproducible methodology for tear profiling studies in the clinical monitoring of patients. © 2012 Molecular Vision. Source

Garcia I.,Bioftalmik | Etxebarria J.,Hospital de Cruces | Boto-De-Los-Bueis A.,Hospital Universitario La Paz | Diaz-Valle D.,Hospital Clinico San Carlos | And 10 more authors.
Ophthalmology | Year: 2012

Purpose: To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription- polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). Design: Transversal, comparative case series. Participants: We studied 59 eyes from 43 patients clinically diagnosed with LSCD. Methods: Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. Main Outcome Measures: Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. Results: Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. Conclusions: The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/μl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. © 2012 American Academy of Ophthalmology. Source

Garcia I.,Bioftalmik | Etxebarria J.,Cruces Hospital | Merayo-Lloves J.,University of Oviedo | Torras J.,Hospital Clinic de Barcelona | And 5 more authors.
Investigative Ophthalmology and Visual Science | Year: 2013

Purpose. To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis. Methods. We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls. Results. Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively. Conclusions. Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions. © 2013 The Association for Research in Vision and Ophthalmology, Inc. Source

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