Lanthanide Chelates as Donors in Fluorescence Resonance Energy Transfer: Exciting Prospects for Bioaffinity Assay Detection

Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer from a fluorescent donor molecule to an appropriate acceptor molecule and a commonly used technique to develop homogeneous assays. If the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, FRET might occur. As a consequence, the emission of the donor is decreased and the emission of the acceptor (if fluorescent) increased. Furthermore, the distance between the donor and the acceptor needs to be short enough, commonly 10-100 Å. Typically, the close proximity between the donor and the acceptor is achieved via bioaffinity interactions e.g. antibody binding antigen. Large variety of donors and acceptors exist. The selection of the donor/acceptor pair should be done not only based on the requirements of FRET but also the performance expectancies and the objectives of the application should be considered. In this study, the exceptional fluorescence properties of the lanthanide chelates were employed to develop two novel homogeneous immunoassays: a non-competitive hapten (estradiol) assay based on a single binder and a dual-parametric total and free PSA assay. In addition, the quenching efficiencies and energy transfer properties of various donor/acceptor pairs were studied. The applied donors were either europium(III) or terbium(III) chelates; whereas several organic dyes (both fluorescent and quenchers) acted as acceptors. First, it was shown that if the interaction between the donor/acceptor complexes is of high quality (e.g. biotin-streptavidin) the fluorescence of the europium(III) chelate could be quenched rather efficiently. Furthermore, the quenching based homogeneous non-competitive assay for estradiol had significantly better sensitivity (~67 times) than a corresponding homogeneous competitive assay using the same assay components. Second, if the acceptors were chosen to emit at the emission minima of the terbium(III) chelate, several acceptor emissions could be measured simultaneously without significant cross-talk from other acceptors. Based on these results, the appropriate acceptors were chosen for the dual-parameter assay. The developed homogeneous dual-parameter assay was able to measure both total and free PSA simultaneously using a simple mix and measure protocol. Correlation of this assay to a heterogeneous single parameter assay was excellent (above 0.99 for both) when spiked human plasma samples were used. However, due to the interference of the sample material, the obtained concentrations were slightly lower with the homogeneous than the heterogeneous assay, especially for the free PSA. To conclude, in this work two novel immunoassay principles were developed, which both are adaptable to other analytes. However, the hapten assay requires a rather good antibody with low dissociation rate and high affinity; whereas the dual-parameter assay principle is applicable whenever two immunometric complexes can form simultaneously, provided that the requirements of FRET are fulfilled.

Size dependent fluorescence spectroscopy of nanocolloids of ZnO

In this article we present size dependent spectroscopic observations of nanocolloids of ZnO. ZnO is reported to show two emission bands, an ultraviolet (UV) emission band and another in the green region. Apart from the known band gap 380 nm and impurity 530 nm emissions, we have found some peculiar features in the fluorescence spectra that are consistent with the nanoparticle size distribution. Results show that additional emissions at 420 and 490 nm are developed with particle size. The origin of the visible band emission is discussed. The mechanism of the luminescence suggests that UV luminescence of ZnO colloid is related to the transition from conduction band edge to valence band, and visible luminescence is caused by the transition from deep donor level to valence band due to oxygen vacancies and by the transition from conduction band to deep acceptor level due to impurities and defect states. A correlation analysis between the particle size and spectroscopic observations is also discussed.

Biocompatible in-tube solid phase microextraction coupled with liquid chromatography-fluorescence detection for determination of interferon alpha in plasma samples

The present work demonstrates the successful application of automated biocompatible in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC) for determination of interferon alpha(2a) (IFN alpha(2a)) in plasma samples for therapeutic drug monitoring. A restricted access material (RAM, protein-coated silica) was employed for preparation of a lab-made biocompatible in-tube SPME capillary that enables the direct injection of biological fluids as well as the simultaneous exclusion of macromolecules by chemical diffusion barrier and drug pre-concentration. The in-tube SPME variables, such as sample volume, draw/eject volume, number of draw-eject cycles, and desorption mode were optimized, to improve the sensitivity of the proposed method. The IFN alpha(2a) analyses in plasma sample were carried out within 25 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range, from 0.06 to 3.0 MIU mL(-1), with correlation coefficient equal to 0.998. The interday precision of the method presented coefficient of variation lower than 8%. The proposed automated method has adequate analytical sensitivity and selectivity for determination of IFN alpha(2a) in plasma samples for therapeutic drug monitoring. (C) 2010 Elsevier B.V. All rights reserved.

The Influence of Pit and Fissure Sealants on Infrared Fluorescence Measurements

The aim of this in vitro study was to evaluate the influence of pit and fissure sealants on fluorescence readings using lasers. We selected 166 permanent molars and randomly divided them into 4 groups which were each treated with a different sealant (a commercially available clear sealant, 2 opaque sealants and an experimental nanofilled clear sealant). The teeth were independently measured twice by 2 experienced dentists using conventional laser fluorescence (LF) and a laser fluorescence pen device (LFpen), before and after sealing, and again after thermocycling to simulate the thermal stressing between the tooth and the dental materials. Friedman test showed no statistically significant changes using LF and LFpen for the commercial clear sealant group, although values tended to increase after sealing. However, the values increased significantly after thermocycling. There was a statistically significant decrease in fluorescence after application of opaque sealants. After application of the experimental nanofilled clear sealant, LF values increased only after thermocycling, whereas the LFpen values increased after sealing and after thermocycling as well. The intraclass correlation coefficient ranged from 0.87 to 0.96 for interexaminer and 0.82 to 0.94 for intraexaminer reproducibility. It was shown that pit and fissure sealants influence LF and LFpen readings, with the values increasing or decreasing according to the material used. In conclusion, both laser fluorescence devices could be useful as an adjunct to detect occlusal caries under unfilled clear sealants. Nevertheless, surfaces sealed with clear nanofilled material could be assessed using only the LF device. Copyright (C) 2008 S. Karger AG, Basel.

Performance of fluorescence methods, radiographic examination and ICDAS II on occlusal surfaces in vitro

This study compared the performance of fluorescence-based methods, radiographic examination, and International Caries Detection and Assessment System (ICDAS) II on occlusal surfaces. One hundred and nineteen permanent human molars were assessed twice by 2 experienced dentists using the laser fluorescence (LF and LFpen) and fluorescence camera (FC) devices, ICDAS II and bitewing radiographs (BW). After measuring, the teeth were histologically prepared and assessed for caries extension. The sensitivities for dentine caries detection were 0.86 (FC), 0.78 (LFpen), 0.73 (ICDAS II), 0.51 (LF) and 0.34 (BW). The specificities were 0.97 (BW), 0.89 (LF), 0.65 (ICDAS II), 0.63 (FC) and 0.56 (LFpen). BW presented the highest values of likelihood ratio (LR)+ (12.47) and LR- (0.68). Rank correlations with histology were 0.53 (LF), 0.52 (LFpen), 0.41 (FC), 0.59 (ICDAS II) and 0.57 (BW). The area under the ROC curve varied from 0.72 to 0.83. Inter- and intraexaminer intraclass correlation values were respectively 0.90 and 0.85 (LF), 0.93 and 0.87 (LFpen) and 0.85 and 0.76 (FC). The ICDAS II kappa values were 0.51 (interexaminer) and 0.61 (intraexaminer). The BW kappa values were 0.50 (interexaminer) and 0.62 (intraexaminer). The Bland and Altman limits of agreement were 46.0 and 38.2 (LF), 55.6 and 40.0 (LFpen) and 1.12 and 0.80 (FC), for intra- and interexaminer reproducibilities. The posttest probability for dentine caries detection was high for BW and LF. In conclusion, LFpen, FC and ICDAS II presented better sensitivity and LF and BW better specificity. ICDAS II combined with BW showed the best performance and is the best combination for detecting caries on occlusal surfaces. Copyright (C) 2008 S. Karger AG, Basel.

In vitro comparison of laser fluorescence performance with visual examination for detection of occlusal caries in permanent and primary molars

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interactions between the non-ionic surfactant C(12)E(5) and poly(ethylene oxide) studied using dynamic light scattering and fluorescence quenching

Dynamic light scattering measurements have been made to elucidate changes in the coil conformation of a high molecular weight poly(ethylene oxide) (PEG) fraction when the non-ionic surfactant C(12)E(5) is present in dilute solutions. The measurements were made at 20 degrees C as functions of(a) the C(12)E(5) concentration at constant PEO concentration, (b) the PEO concentration at constant C(12)E(5) concentration, and (c) the C(12)E(5)/PEO concentration ratio. The influence of temperature on the interactions in terms of the relaxation time distributions was also examined up to the cloud point. It was found that when the C(12)E(5)/PEO weight ratio was >2 and when the temperature was >14 degrees C, the correlation functions became bimodal with well-separated components. The fast mode derives fi om individual surfactant micelles which are present in the solution at high number density. The appearance of the slow mode, which dominates the scattering, is interpreted as resulting from the formation of micellar clusters due to an excluded-volume effect when the high molar mass (M = 6 x 10(5)) PEO is added to the surfactant solution. It is shown that the micellar clusters form within the PEO coils and lead to a progressive swelling of the latter for steric reasons. The dimensions of the PEO/C(12)E(5) complex increase with increasing surfactant concentration to a value of R(H) approximate to 94 nm (R(g) approximate to 208 nm) at C-C12E5 = 3.5%. Fluorescence quenching measurements show that the average aggregation number of C(12)E(5) increases significantly on addition of the high molar mass PEG. With increasing temperature toward the cloud point the clusters increase in number density and/or become larger. The cloud point is substantially lower than that for C12E5 in water solution and is strongly dependent on the PEO concentration.

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: Electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4- carboxylic acid (TOAC). TOAC replaced Asp 1 (TOAC 1-AII) and Val 3 (TOAC 3-AII) in AII and was inserted prior to Arg 1 (TOAC 0-BK) and replacing Pro 3 (TOAC 3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (TC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, τ C increased due to viscosity effects. Calculation of τ Cpeptide/τ CTOAC ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC 1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC 3 derivatives acquired more restricted conformations. Fluorescence spectra of All and its derivatives were especially sensitive to the ionization of Tyr 4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC 3-AII The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation. © 2004 Wiley Periodicals, Inc.

In vitro assessment of dentin erosion after immersion in acidic beverages: Surface profile analysis and energy-dispersive X-ray fluorescence spectrometry study

The aim of this study was to investigate the effects of some acidic drinks on dentin erosion, using methods of surface profile (SP) analysis and energy-dispersive X-ray fluorescence spectrometry (EDXRF). One hundred standardized dentin slabs obtained from bovine incisor roots were used. Dentin slabs measuring 5x5 mm were ground flat, polished and half of each specimen surface was protected with nail polish. For 60 min, the dentin surfaces were immersed in 50 mL of 5 different drinks (Gatorade®, Del Valle Mais orange juice®, Coca-Cola®, Red Bull® and white wine), 20 blocks in each drink. The pH of each beverage was measured. After the erosive challenge, the nail polish was removed and SP was analyzed. The mineral concentration of dentin surfaces was determined by means of EDXRF. Data were analyzed statistically by ANOVA and Tukey's test (α=0.05). SP analysis showed that Red-Bull had the highest erosive potential (p<0.05). EDXRF results exhibited a decrease in phosphate in the groups immersed in Red-Bull, orange juice and white wine (p<0.05), and no significant difference in calcium content between the reference surface and eroded surface. In conclusion, this study demonstrated that all studied beverages promoted erosion on root dentin and Red Bull had the highest erosive potential. There was no correlation between pH of beverages and their erosive potential and only the P content changed after erosive challenge.

Dental fluorescence: Potential forensic use

In cases of identification of bones, skeletal segments or isolated bones, searching for biotypologic diagnostic data to estimate an individual's age enables comparing these data with those of missing individuals. Enamel, dentin and pulp undergo remarkable changes during an individual's life. The enamel becomes more mineralized, smoother and thinner, and deteriorates because of physiological and pathological factors. Dental pulp decreases in volume due to the deposition of secondary dentin; thus, the dentin becomes thicker with time. In natural teeth, the fluorescence phenomenon occurs in dentin and enamel and changes in those tissues may alter the expression of the natural tooth color. The aim of this study was to assess the correlation between age and teeth fluorescence for individuals from different age groups. The sample consisted of 66 randomly selected Brazilians of both genders aged 7-63 years old. They were divided into 6 groups: Group 1 - aged 7-12 years, Group 2 - aged 13-20 years, Group 3 - aged 21-30 years, Group 4 - aged 31-40 years, Group 5 - aged 41-50 years and Group 6 - aged between 51 and 63 years. Upper right or left central incisors were used for the study. Restored and aesthetic rehabilitated teeth were excluded from the sample. The measurement of tooth fluorescence was carried out via computer analysis of digital images using the software ScanWhite DMC/Darwin Systems - Brazil. It was observed that dental fluorescence decreases when comparing the age groups 21-30, 31-40, 41-50 and 51-63 years. The results also showed that there is a statistically significant difference between the groups 41-50 years and 21-30 years (p=. 0.005) and also among the group 51-63 years and all other groups (p< 0.005). It can be concluded that dental fluorescence is correlated with age and has a similar and stable behavior from 7 to 20 years of age. It reaches its maximum expected value at the age of 26.5 years and thereafter decreases. © 2013 Elsevier B.V.

Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro performance of a pen-type laser fluorescence device and bitewing radiographs for approximal caries detection in permanent and primary teeth

To evaluate the performance of a pen‑type laser fluorescence device (DIAGNOdent 2190; LFpen, KaVo, Germany) and bitewing radiographs (BW) for approximal caries detection in permanent and primary teeth. Materials and Methods: A total of 246 anterior approximal surfaces (102 permanent and 144 primary) were selected. Contact points were simulated using sound teeth. Two examiners assessed all approximal surfaces using LFpen and BW. The teeth were histologically assessed for the reference standard. Optimal cut‑off limits were calculated for LFpen for primary and permanent teeth. Sensitivity, specificity, accuracy and area under the receiver operating characteristic curve (Az) were calculated for D1 (enamel and dentin lesions) and D3 (dentin lesions) thresholds. The reproducibility was assessed by intraclass correlation coefficient (ICC) and Cohen's weighted kappa values. Results: For permanent teeth, the LFpen cut‑off were 0– 27 (sound), 28– 33 (enamel caries) and >33 (dentin caries). For primary teeth, the LFpen cut‑off were 0– 7 (sound), 8– 32 (enamelcaries) and >32 (dentin caries). The LFpen presented higher sensitivity values than BW for primary teeth (0.58 vs. 0.32 at D1 and 0.80 vs. 0.47 at D3) and permanent teeth (0.80 vs. 0.57 at D1 and 0.94 vs. 0.51 at D3). Specificity did not show a significant difference between the methods. Rank correlations with histology were 0.59 and 0.83 (LFpen) and 0.36 and 0.70 (BW) for primary and permanent teeth, respectively, considering all lesions. ICC values for LFpen were 0.71 (inter) and 0.86 (intra) for permanent teeth and 0.94 (inter) and 0.90/0.99 for primary teeth. Kappa values for BW were 0.69 (inter) and 0.68/0.90 (intra) for permanent teeth and 0.64 (inter) and 0.89/0.89 for primary teeth. Conclusion: LFpen presented better reproducibility for primary and permanent teeth and higher accuracy in detecting caries lesions at D1 threshold than BW for permanent teeth. LFpen should be used as an adjunct method for approximal caries detection.

Trace elements as tumor biomarkers and prognostic factors in breast cancer: a study through energy dispersive x-ray fluorescence

Background The application and better understanding of traditional and new breast tumor biomarkers and prognostic factors are increasing due to the fact that they are able to identify individuals at high risk of breast cancer, who may benefit from preventive interventions. Also, biomarkers can make possible for physicians to design an individualized treatment for each patient. Previous studies showed that trace elements (TEs) determined by X-Ray Fluorescence (XRF) techniques are found in significantly higher concentrations in neoplastic breast tissues (malignant and benign) when compared with normal tissues. The aim of this work was to evaluate the potential of TEs, determined by the use of the Energy Dispersive X-Ray Fluorescence (EDXRF) technique, as biomarkers and prognostic factors in breast cancer. Methods By using EDXRF, we determined Ca, Fe, Cu, and Zn trace elements concentrations in 106 samples of normal and breast cancer tissues. Cut-off values for each TE were determined through Receiver Operating Characteristic (ROC) analysis from the TEs distributions. These values were used to set the positive or negative expression. This expression was subsequently correlated with clinical prognostic factors through Fisher’s exact test and chi-square test. Kaplan Meier survival curves were also evaluated to assess the effect of the expression of TEs in the overall patient survival. Results Concentrations of TEs are higher in neoplastic tissues (malignant and benign) when compared with normal tissues. Results from ROC analysis showed that TEs can be considered a tumor biomarker because, after establishing a cut-off value, it was possible to classify different tissues as normal or neoplastic, as well as different types of cancer. The expression of TEs was found statistically correlated with age and menstrual status. The survival curves estimated by the Kaplan-Meier method showed that patients with positive expression for Cu presented a poor overall survival (p < 0.001). Conclusions This study suggests that TEs expression has a great potential of application as a tumor biomarker, once it was revealed to be an effective tool to distinguish different types of breast tissues and to identify the difference between malignant and benign tumors. The expressions of all TEs were found statistically correlated with well-known prognostic factors for breast cancer. The element copper also showed statistical correlation with overall survival.

Performance of fluorescence-based and conventional methods of occlusal caries detection in primary molars - an in vitro study

International Journal of Paediatric Dentistry 2012; 22: 459466 Aim. This in vitro study aimed to test the performance of fluorescence-based methods in detecting occlusal caries lesions in primary molars compared to conventional methods. Design. Two examiners assessed 113 sites on 77 occlusal surfaces of primary molars using three fluorescence devices: DIAGNOdent (LF), DIAGNOdent pen (LFpen), and fluorescence camera (VistaProof-FC). Visual inspection (ICDAS) and radiographic methods were also evaluated. One examiner repeated the evaluations after one month. As reference standard method, the lesion depth was determined after sectioning and evaluation in stereomicroscope. The area under the ROC curve (Az), sensitivity, specificity, and accuracy of the methods were calculated at enamel (D1) and dentine caries (D3) lesions thresholds. The intra and interexaminer reproducibility were calculated using the intraclass correlation coefficient (ICC) and kappa statistics. Results. At D1, visual inspection presented higher sensitivities (0.970.99) but lower specificities (0.180.25). At D3, all the methods demonstrated similar performance (Az values around 0.90). Visual and radiographic methods showed a slightly higher specificity (values higher than 0.96) than the fluorescence based ones (values around 0.88). In general, all methods presented high reproducibility (ICC higher than 0.79). Conclusions. Although fluorescence-based and conventional methods present similar performance in detecting occlusal caries lesions in primary teeth, visual inspection alone seems to be sufficient to be used in clinical practice.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

Abstract Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.