Fluorescence Intensity Measurements Research Articles

Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes

Background/Aims: Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. Methods: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. Results: Monensin (10⁻⁵M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (I_Na) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (I_Ca). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (I_Na-Ca) was activated particularly in the reverse mode. Na-K pump current (I_Na-K) was also activated. Notably, the inward rectifying K current (I_K1) was not affected, and the change in the delayed outward K current (I_K) was not evident. Conclusion: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the I_Ca, the activation of the reverse mode of I_Na-Ca, and the increased I_Na-K, and second due to the decreased I_Na. The I_K and the I_K1 may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

Versatile Optofluidic Solid-Core/Liquid-Cladding Waveguide Based on Evanescent Wave Excitation.

In this paper, we present the design and operation of a solid-core/liquid-cladding (SL) waveguide excited by an evanescent wave. To do this, an optical fiber is integrated into a microfluidic channel and pumped along the fiber axis, ensuring the cladding solution is excited by the evanescent field of the guided mode at the core/cladding interface. The pump beam is guided by the total internal reflection in the fiber, providing a uniform excitation along the microfluidic channel. The evanescent wave provides precise excitation to the dye molecules in close proximity to the core/cladding interface, which significantly reduces the background fluorescence and increases the signal-to-noise ratio. Fluorescence intensity measurements of different dye concentrations and refractive indices of the cladding solution are conducted to evaluate their influences on the propagation loss, which shows that the peak intensity propagation loss can be as low as about 0.1 dB/cm. We further exemplify that the intensity of the fluorescence emission and the dye concentration show good linearity when the dye is in the low concentration region (<250 μM). A broad-band and simultaneous light source with a single pump light is also demonstrated by employing cascade SL waveguide segments through fluorescence resonance energy transfer. The proposed SL waveguide demonstrates excellent robustness and is easy to fabricate and use, providing a versatile platform for a variety of applications, such as high-sensitivity detection of low-concentration samples, multiband on-chip light sources, and simultaneous measurement of multiplexed parameters.

Synchronous Spectrofluorimetry Coupled with Third-Order Derivative Signal Processing for the Simultaneous Quantitation of Telmisartan and Chlorthalidone Drug Combination in Human Plasma.

This study is the first to develop and optimize a method for the simultaneous determination of chlorthalidone (CLT) and telmisartan (TEL) in, human plasma samples as well as in their newly released pharmaceutical tablet form, (Telmikind-CT 40®). The method is based on measuring fluorescence intensity, employing synchronous fluorescence mode coupled to third-order derivative signal processing, 0.5% w/v cetyl trimethyl ammonium bromide was used as cationic surfactant to enhance the fluorescence signal intensity and improve method sensitivity. The third-order derivative synchronous spectra of CLT and TEL are well separated with two zero-crossing points which allowed for the determination of CLT and TEL at 362nm and 351nm, respectively. Different experimental parameters were carefully investigated and optimized, calibration curves were constructed over concentration ranges of 20-1200ng.mL-1 and 5-800ng.mL-1 for CLT and TEL respectively. The developed method is simple and rapid, analytical parameters were validated according to ICH guidelines and high sensitivity was achieved as represented by limits of detection (LOD) of 4.69 and 1.58ng.mL-1 for CLT and TEL respectively.

Chip‐based duplex real‐time PCR for water quality monitoring concerning Legionella pneumophila and Legionella spp.

AbstractBased on biomolecular methods, rapid and selective identification of human pathogenic water organisms becomes an important issue. Legionella spp., are pathogenic water bacteria with worldwide significance. Prevalent detection methods for these microorganisms are time and/or cost intensive. We describe a detection setup and relating DNA assay. A miniaturized real‐time polymerase chain reaction (real‐time PCR) for direct on‐line discrimination of Legionella pneumophila and Legionella spp. was established and integrated into a real‐time PCR‐chip‐system. The PCR‐chip device combines a temperature controlling unit and a fluorescence intensity measurement. It was designed to achieve rapid amplification, using an approach of real‐time fluorescence read out with the intercalating dye EvaGreen® and melting curve analysis, without requiring multiple probes. The presented results exhibit reproducibility and good sensitivity, showing that the setup is suitable for robust, rapid and cost‐efficient detection and monitoring of a variety of Legionella spp.in urban water samples.

Chemical structure and inhibition on α-glucosidase of polysaccharides from corn silk by fractional precipitation

Aimed to explore different corn silk polysaccharide (CSP) fractions derived by ethanol precipitation on the physicochemical properties and biological activities, four fractions (CSP20, CSP40, CSP60, and CSP80) were obtained. CSPs consisted of mannose, galactose, arabinose, rhamnose, xylose, and glucose with different ratios, and exhibited different total sugar content, uronic acid content, protein content, and total phenols content. All fractions also showed different physical properties, such as molecular weight, intrinsic viscosity, particle size, and microstructure. Besides, CSP80 exhibited stronger antioxidant activity and α-glucosidase inhibitory activity than the other three fractions. Enzyme kinetic analysis suggested that CSP80 inhibited α-glucosidase by mixed type and reversible mechanisms, respectively. Fluorescence intensity measurements confirmed that the secondary structure of α-glucosidase was changed by the binding of CSP80. Isothermal titration calorimetry (ITC) results illustrated that the binding of CSP80 to α-glucosidase complex was spontaneous driven by enthalpy and hydrogen bonds played a major role in the binding.

Quantitative Fundus Autofluorescence in HCQ Retinopathy.

PurposeTo increase our understanding of the mechanisms underlying hydroxychloroquine (HCQ) retinopathy, analyses by quantitative fundus autofluorescence (qAF) and near-infrared fundus autofluorescence (NIR-AF) were compared to results obtained by recommended screening tests.MethodsThirty-one patients (28 females, 3 males) were evaluated with standard automated perimetry and spectral domain optical coherence tomography (SD-OCT); 28 also had multifocal electroretinography (mfERG). Measurement of short-wavelength fundus autofluorescence (SW-AF) by qAF involved the use of an internal fluorescent reference and intensity measurements in eight concentric segments at 7° to 9° eccentricity. For semiquantitative analysis of NIR-AF, intensities were acquired along a vertical axis through the fovea.ResultsFour of 15 high-dose (total dose >1000 g, daily dose >5.0 mg/kg) patients and one of 16 low-dose (total dose <1000 g, daily dose 4.4 mg/kg) patients were diagnosed with HCQ-associated retinopathy based on abnormal 10-2 visual fields, SD-OCT, and SW-AF imaging. Three of the high-dose patients also had abnormal mfERG results. Of the five patients exhibiting retinopathy, two had qAF color-coded images revealing higher intensities inferior, nasal, and lateral to the fovea. The abnormal visual fields also exhibited superior-inferior differences. Mean NIR-AF gray-level intensities were increased in four high-dose patients with no evidence of retinopathy. In two patients with retinopathy, NIR-AF intensity within the parafovea was below the normal range. One high-dose patient (6.25 mg/kg) had only abnormal mfERG results.ConclusionsThese findings indicate that screening for HCQ retinopathy should take into consideration superior-inferior differences in susceptibility to HCQ retinopathy.

Fluorescence intensity and lifetime redox ratios detect metabolic perturbations in T cells.

The auto-fluorescent coenzymes reduced nicotinamide dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) allow label-free detection of cellular metabolism. The optical redox ratio, which is traditionally computed as the ratio of NADH and FAD intensities, allows quantification of cell redox state. In addition to multiple formulations of the optical redox ratio from NADH and FAD intensity measurements, a fluorescence lifetime redox ratio (FLIRR) based on the fractions of protein-bound NADH and FAD was developed to overcome the limitations of experimental factors that influence fluorescence intensity measurements. In this paper, we compare fluorescence-intensity computations of the optical redox ratio with the fluorescence lifetime redox ratio for quiescent and activated T cells. Fluorescence lifetime images of NAD(P)H and FAD of T cells were acquired with a two-photon fluorescence lifetime microscope. Metabolic perturbation experiments, including inhibition of glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid synthesis revealed differences between the intensity and lifetime redox ratios. Statistical analysis reveals that the FLIRR has a lower standard deviation and skewness (two-tail T-test, P value = 0.05) than the intensity redox ratio. Correlation analysis revealed a weak relationship between FLIRR and intensity redox ratio for individual cells, with a stronger correlation identified for activated T cells (Linear regression, R-value = 0.450) than quiescent T cells (R-value = 0.172). Altogether, the results demonstrate that while both the fluorescence lifetime and intensity redox ratios resolve metabolic perturbations in T cells, the endpoints are influenced by different metabolic processes.

Complex Analysis of Fluorescence Intensity Fluctuations of Molecular Compounds

A method is proposed for the complex analysis of fluctuations in the fluorescence intensity of molecular compounds, which allows determining the structural composition of protein oligomers. The idea of the method is to analyze the photon counting histograms of experimental measurements using principal component analysis to assess the presence of oligomeric compounds, and to perform hierarchical cluster analysis, to determine the data classes corresponding to various molecular compounds, followed by selecting cluster medoids to determine the oligomeric composition of protein complexes. The efficiency of the analysis algorithms developed within the framework of the proposed method was confirmed on simulated and experimental photon counting histograms of the measured fluorescence intensity fluctuations of monomeric and dimeric forms of green-fluorescent protein (GFP).

COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids

BackgroundSignificant progress has been made in cell replacement therapy for neural retinal diseases using retinal cells differentiated from human pluripotent stem cells. Low tumorigenicity and the ability to mature to form synaptic junctions make precursor cells a promising donor source. Here, we attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs).MethodsA CRX-tdTomato-tagged hESC line was generated to track retinal precursors in 3D retinal organoids. COCO, a multifunctional antagonist of the Wnt, TGF-β, and BMP pathways, was employed to 3D organoid differentiation schemes for enhanced photoreceptor precursor cells. Organoid fluorescence intensity measurement was used to monitor retinalization tendency with the number of precursors further checked by flow cytometry. Signature gene expression during organoid differentiation were assessed by qPCR and immunocytochemistry after COCO supplementation.ResultsCRX-positive cells can be spatiotemporally tracked by tdTomato without affecting retinalization during retinal organoid differentiation. Fluorescence intensity of organoids, which turned out highly consistent with flow cytometry measurement, allowed us to determine the differentiation efficiency of precursors during organoid culturing directly. Using COCO as an auxiliary supplement, rather than alone, can yield an increased number of photoreceptor precursors in the early stage of organoid differentiation. Over a longer time-frame, photoreceptor precursors enhanced their fate of cones and decreased fate of rods after treatment with COCO.ConclusionsTracing with the CRX-reporter system showed that in retinal organoids derived from human pluripotent stem cells, COCO increased the differentiation efficiency of photoreceptor precursors and cones.

Abstract 5384: Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients

Abstract Circulating tumor cells (CTCs) can provide information on drug target expression, response to therapy, and disease prognosis from a non-invasive blood draw. Currently, investigating biomarkers on CTCs is difficult due to challenges of developing multiplexed assays that also identify rare cells. Presence of the androgen receptor splice variant ARv7 in prostate cancer cells is associated with resistance to second generation anti-androgen therapies. We report here the analytical validation of an immunofluorescence assay for characterization of ARv7 protein expression on CTCs using the RareCyte platform - an end-to-end platform that combines CTC sample preparation, multiparameter fluorescence staining, digital imaging, and single cell retrieval. Blood samples spiked with positive and negative cell lines for ARv7 expression were processed using the AccuCyte Sample Preparation System. Slides were auto-stained by immunofluorescence with the RarePlex ARv7 CTC Panel Kit comprised of a three-channel CTC detection base plus an ARv7 biomarker channel. The detection base consists of a nuclear dye, anti-CD45 antibody to exclude white blood cells, and cocktailed antibodies to cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM). Stained slides were imaged with the CyteFinder Instrument. CTCs were identified using machine learning-based algorithms and confirmed by user review. Mean fluorescence intensity (MFI) measurements were used as a metric for ARv7 expression on confirmed CTCs. Analytic validation studies of the AR-V7 CTC assay were performed using 22RV1 (high), LNCaP (low), and BT-474 (negative) cell lines. Performance characteristics tested for ARv7 included accuracy, sensitivity, specificity, repeatability, and inter-stainer run coefficient of variation. Performance metrics for CTC recovery were calculated on spike-in and clinical samples. For recovery calculations, the number of CTCs found with the ARv7 assay was compared to the number of CTCs found with the CTC detection base assay. An ARv7 MFI threshold that segregated negative and positive cell lines was statistically defined. This threshold identified 80% of 22RV1 cells as positive for ARv7, 97% of BT-474 cells as negative, with an overall accuracy of 90%. When the assay was applied to clinical prostate cancer samples, staining with proper nuclear localization was observed. CTC recovery was at least as high with the ARv7 assay as with the base CTC detection assay. Citation Format: Daniel Campton, Edward Lo, Lillian Costandy, Brady Gardner, Ryan Houston, Jeffery L. Werbin, Kyla Teplitz, Daniel E. Sabath, Alisa C. Clein, Celestia S. Higano, Tanisha M. Mojica, Kristin Province, Eric P. Kaldjian, Arturo B. Ramirez, Tad George. Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5384.

Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore's inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695-707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination.IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Correlations between functional porphyrin positions and accumulation in cancer cells

Porphyrin is accumulated in tumours due to its interaction with protein. Cancer therapy with porphyrin as a carrier molecule is attracting attention. Porphyrin displays two functional sites termed β- and meso-positions. A correlation between the functional position on the porphyrin molecule and the ability to accumulate in cancer cells is observed in the present study. The accumulation of porphyrin derivatives was determined by measuring fluorescence intensity after incubation for 2 and 24 h. The accumulation of cancer cells depended on the position and length of functional groups. Estimated binding constants between porphyrin and bovine serum albumin suggest that the position of functional groups leads to changes in binding affinity and influences the accumulation of porphyrin derivatives in cancer cells.

Bleaching correction for DNA measurements in highly diluted solutions using confocal microscopy.

Determining the concentration of nucleic acids in biological samples precisely and reliably still is a challenge. In particular when only very small sample quantities are available for analysis, the established fluorescence-based methods give insufficient results. Photobleaching is seen as the main reason for this. In this paper we present a method to correct for the photobleaching effect. Using confocal microscopy with single molecule sensitivity, we derived calibration curves from DNA solutions with defined fragment length. We analyzed dilution series over a wide range of concentrations (1 pg/μl-1000 pg/μl) and measured their specific diffusion coefficients employing fluorescence correlation spectroscopy. Using this information, we corrected the measured fluorescence intensity of the calibration solutions for photobleaching effects. We evaluated our method by analyzing a series of DNA mixtures of varying composition. For fragments smaller than 1000 bp, our method allows to determine sample concentrations with high precision in very small sample quantities (< 2 μl with concentrations < 20 pg/μl). Once the technical parameters are determined and remain stable in an established process, our improved calibration method will make measuring molecular biological samples of unknown sequence composition more efficient, accurate and sample-saving than previous methods.

Assessment of the impact of horse-chestnut leaf miner (Cameraria ohridella Deschka &amp; Dimič, 1986) on photosynthesis of common horse chestnut (Aesculus hippocastanum Linnaeus, 1753)

The article presents the results of studies on Cameraria ohridella Deschka &amp; Dimič, 1986 feeding on the state of the photosynthetic apparatus in Aesculus hippocastanum Linnaeus, 1753. The studies were carried out in June 2019 (during the life of C. ohridella first generation) on the territory of the Botanical Garden of the Oles Honchar Dnipro National University. Photosynthesis is one of the processes most susceptible to stress factors; important information on state of the photosynthetic apparatus in plants under influence of various abiotic and biotic factors can be obtained by fluorescence analysis. Chlorophyll fluorescence is an indicator that allow to investigate a behavior of photochemical reactions related to system II (PSII) in living objects as an indicator the most sensitive to effect of environmental factors. One of the effective methods for plant state monitoring is a technique on chlorophyll fluorescence induction (CFI) measurement, which at the present stage can be introduced due to the use of biosensors. The objective of our study was to establish characteristics of C. ohridella vital activity on the photosynthesis process in A. hippocastanum trees using CFI evaluation technique. To diagnose photosynthesis violation of native chlorophyll in a living leaf of A. hippocastanum, we used a portable fluorometer “Floratest” (spectral range for measuring fluorescence intensity 670 to 770 nm) developed by V. M. Glushkov Institute of Cybernetics of National Academy of Sciences of Ukraine. The studies were conducted using alive A. hippocastanum leaves not damaged by C. ohridella and, accordingly, the damaged ones. Analysis of the data obtained showed that the worm feeding affects all CFI parameters (initial CFI value (non-damaged leaf: 582 relative units, damaged: 264); “plateau” CFI value (1460 to 722); maximum CFI (1890 to 940) and stationary CFI value after light adaptation of the leaf (1530 to 746)). Our studies have shown that CFI technique using “Floratest” apparatus allow express determining the general condition of the plant by assessing the main process of vital activity, namely photosynthesis. Analysis of chlorophyll fluorescence parameters is a powerful and effective tool for determining the phytophage effect on the plant.

Molecular Phenotyping of White Striping and Wooden Breast Myopathies in Chicken.

The White Striping (WS) and Wooden Breast (WB) defects are two myopathic syndromes whose occurrence has recently increased in modern fast-growing broilers. The impact of these defects on the quality of breast meat is very important, as they greatly affect its visual aspect, nutritional value, and processing yields. The research conducted to date has improved our knowledge of the biological processes involved in their occurrence, but no solution has been identified so far to significantly reduce their incidence without affecting growing performance of broilers. This study aims to follow the evolution of molecular phenotypes in relation to both fast-growing rate and the occurrence of defects in order to identify potential biomarkers for diagnostic purposes, but also to improve our understanding of physiological dysregulation involved in the occurrence of WS and WB. This has been achieved through enzymatic, histological, and transcriptional approaches by considering breast muscles from a slow- and a fast-growing line, affected or not by WS and WB. Fast-growing muscles produced more reactive oxygen species (ROS) than slow-growing ones, independently of WS and WB occurrence. Within fast-growing muscles, despite higher mitochondria density, muscles affected by WS or WB defects did not show higher cytochrome oxidase activity (COX) activity, suggesting altered mitochondrial function. Among the markers related to muscle remodeling and regeneration, immunohistochemical staining of FN1, NCAM, and MYH15 was higher in fast- compared to slow-growing muscles, and their amount also increased linearly with the presence and severity of WS and WB defects, making them potential biomarkers to assess accurately their presence and severity. Thanks to an innovative histological technique based on fluorescence intensity measurement, they can be rapidly quantified to estimate the injuries induced in case of WS and WB. The muscular expression of several other genes correlates also positively to the presence and severity of the defects like TGFB1 and CTGF, both involved in the development of connective tissue, or Twist1, known as an inhibitor of myogenesis. Finally, our results suggested that a balance between TGFB1 and PPARG would be essential for fibrosis or adiposis induction and therefore for determining WS and WB phenotypes.

Fluorescence spectroscopy for determination of dapagliflozin in pure form and its tablets formulation; Application to content uniformity testing.

A new, valid method was developed for quantitative spectrofluorimetric analysis of dapagliflozin (DGF) in its pure form and its commercially available tablets (Farxiga®). The proposed analytical method based on measurement of fluorescence intensity for DGF at 303 nm after excitation at 278 nm. Various experimental parameters influencing the fluorescence of DGF were examined to produce the optimal conditions. DGF was successively assayed in concentration range of (100-1000 ng mL-1) with lower detection limit (LOD) of 26.49 and quantitation limit (LOQ) was 79.48 ng mL-1. The suggested method was validated according to ICH guidelines for the estimation of DGF in its pure form and its new dosage form with percent recovery of 100.43 ± 1.15. The proposed method was adapted to examine DGF in content uniformity testing according to United States Pharmacopeia guidelines. This method can be used in routine analysis of DGF in quality control lab.

Dual sensitivity enhancement in gold nanoparticle-based lateral flow immunoassay for visual detection of carcinoembryonic antigen.

In this work, we presented the development of cost-effectivedual sensitivity enhancement in gold nanoparticle-based lateral flow test strip for detection of carcinoembryonic antigen. On the one hand, we employed protein G as a host matrix for oriented immobilization of antibodies within the nitrocellulose membrane. On the other hand, we utilized gold enhancement approach to visualize the final signals effectively. Primary examinations revealed that the smaller sized nanoparticles have greater signal enhancement compared to bigger ones. So, mono-dispersed gold nanoparticles with average diameters of 11.40 ± 1.40nm were utilized as tags. The measurement of fluorescent intensity of FITC-tagged secondary antibody attached to the polyclonal antibody, in the presence/absence of protein G as a host matrix on microplate wells, showed the successful oriented immobilization of antibodies via the host matrix. The FESEM images confirmed the attachment and growth of nanoparticles within the porous nitrocellulose membrane, after gold enhancement. Finally, under the optimized conditions, the developed strip could quantify the standard values of target within 2-50ng/mL range with a limit of detection of 0.35ng/mL. This strategy enabled the reduction of antibody consumption from a conventional amount of 0.6µg/strip down to 0.012µg/strip. The serum samples containing carcinoembryonic antigen were also successfully analyzed by the developed strip with a visual detection limit of 10ng/mL, which confirms favorable characteristics of the developed test strip for point-of-care applications.

Influence of metals and metalloids on the composition and fluorescence quenching of the extracellular polymeric substances produced by the polymorphic fungus Aureobasidium pullulans

Aureobasidium pullulans is a ubiquitous and widely distributed fungus in the environment, and exhibits substantial tolerance against toxic metals. However, the interactions between metals and metalloids with the copious extracellular polymeric substances (EPS) produced by A. pullulans and possible relationships to tolerance are not well understood. In this study, it was found that mercury (Hg) and selenium (Se), as selenite, not only significantly inhibited growth of A. pullulans but also affected the composition of produced EPS. Lead (Pb) showed little influence on EPS yield or composition. The interactions of EPS from A. pullulans with the tested metals and metalloids depended on the specific element and their concentration. Fluorescence intensity measurements of the EPS showed that the presence of metal(loid)s stimulated the production of extracellular tryptophan-like and aromatic protein-like substances. Examination of fluorescence quenching and calculation of binding constants revealed that the fluorescence quenching process for Hg; arsenic (As), as arsenite; and Pb to EPS were mainly governed by static quenching which resulted in the formation of a stable non-fluorescent complexes between the EPS and metal(loid)s. Se showed no significant interaction with the EPS according to fluorescence quenching. These results provide further understanding of the interactions between metals and metalloids and EPS produced by fungi and their contribution to metal(loid) tolerance.Key points• Metal(loid)s enhanced production of tryptophan- and aromatic protein-like substances.• Non-fluorescent complexes formed between the EPS and tested metal(loid)s.• EPS complexation and binding of metal(loid)s was dependent on the tested element.• Metal(loid)-induced changes in EPS composition contributed to metal(loid) tolerance.

Cell-Based No-Wash Fluorescence Assays for Compound Screens Using a Fluorescence Cytometry Plate Reader.

High-throughput cell-based fluorescent imaging assays often require removal of background fluorescent signal to obtain robust measurements. Processing high-density microplates to remove background signal is challenging because of equipment requirements and increasing variation after multiple plate wash steps. Here, we present the development of a wash-free cell-based fluorescence assay method for high-throughput screening using a laser scanning fluorescence plate cytometer. The cytometry data consisted of cell count and fluorescent intensity measurements for phenotypic screening. We obtained robust screening results by applying this assay methodology to the lysosomal storage disease Niemann-Pick disease type A. We further demonstrated that this cytometry method can be applied to the detection of cholesterol in Niemann-Pick disease type C. Lastly, we used the Mirrorball method to obtain preliminary results for the detection of Zika and Dengue viral envelope protein. The advantages of this assay format include 1) no plate washing, 2) 4-fold faster plate scan and analysis time, 3) high throughput, and 4) >10-fold smaller direct data files. In contrast, traditional imaging assays require multiple plate washes to remove the background signal, long plate scan and data analysis times, and large data files. Therefore, this versatile and broadly applicable Mirrorball-based method greatly improves the throughput and data quality of image-based screening by increasing sensitivity and efficiency while reducing assay artifacts.SIGNIFICANCE STATEMENTThis work has resulted in the development of broadly applicable cell-based fluorescence imaging assays without the requirement of washing out reagents to reduce background signal, which effectively decreases the need for extensive plate processing by the researcher. We demonstrate this high-throughput method for drug screening against lysosomal storage diseases and a commonly used viral titer assay.

ICG-induced NIR fluorescence mapping in patients with head & neck tumors after the previous radiotherapy.

The distinction between tumor and healthy tissues is complicated in the areas previously subjected to radiation therapy (RT). This is related to the fact that tissues can undergo delayed and irreversible deterioration such as inflammation, vascular alteration and fibrosis. The trials related to the fluorescence -guided surgery (FSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) patients, previously subjected to RT, have not yet been reported. The present study addresses for the first time the possibilities of tumor near-infrared (NIR) imaging using Indocynaine Green (ICG) in irradiated areas. Four patients with histologically confirmed HNSCC were included in this study. All included patients were previously treated with RT with at least 50 Gy. RT-radiation fields from original treatment fully encompassed the second tumor or recurrence. ICG was injected via cephalic vein 45 min before the images were captured using a NIR camera system Artemis. The images were also captured before ICG injection serving as background signal. The fluorescence intensity measurements were carried out using specially designed software. ICG fluorescence clearly demonstrated a significant difference in fluorescence intensity between healthy and tumor tissues in 2 of 4 patients. Histology post-resection analysis confirmed a complete tumor resection with safe surgical margins. No difference between tumor and surrounding healthy tissue was detected in patients with an epidermoid carcinoma developed from sclerohypertrophic lichen. In our pilot study, we clearly established the feasibility of using NIR FGS with ICG to delineate tumor and healthy tissues in irradiated areas in infiltrating lichen-free tumors.