Fluorescence-based Detection System Research Articles

A novel LD-targeting cysteine-activated fluorescent probe for diagnosis of APAP-induced liver injury and its application in food analysis

Cysteine (Cys) not only plays an indispensable role in maintaining the redox balance in organisms, but is also an important nutrient in the food industry. Fluorescence-based detection systems have emerged as an effective method to track the locations and concentrations of different species. To achieve efficient monitoring of Cys in both food samples and biological systems, a novel lipid droplet (LD) targeted fluorescent probe (namely NIT-Cys) was constructed for the turn-on detection of Cys, characterized by a large Stokes shift (142 nm), a short response time (<8 min), and a low Cys detection limit (39 nM). Furthermore, the NIT-Cys probe has been successfully used not only to quantify the amounts of Cys in selected food samples, but also to enable the visualization of endogenous Cys in acetaminophen (APAP)-induced drug-induced liver injury cells, zebrafish larvae and mice models. Consequently, the work presented here provides an efficient tool for monitoring Cys.

Detection of Mycobacterium tuberculosis Complex Genetic Material in a Free-Living Brown Bear (Ursus arctos).

Mycobacterium tuberculosis complex (MTBC) has rarely been detected in bears (Ursidae). We describe detection of MTBC genetic material using a single-tube, high-multiplex PCR and fluorescence-based detection system in a throat swab collected from a free-living, problem individual during immobilization and telemetry collar deployment. Mycobacterial culture was negative in all samples.

A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine

BackgroundAnti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely...

Small Molecule p53-MDM2 Inhibitors As Potential Regulators of p53-Dependent Genes Expression in Multiple Sclerosis

Transcription factor p53 plays an important role in numerous processes such as apoptosis, cell cycle regulation, metabolism, etc. This protein is believed to regulate apoptosis and neuronal death in various disorders such as multiple sclerosis (MS), Parkinson's and Alzheimer's diseases. In many cases p53 inactivation is caused by overexpression of its negative regulator - E3 ubiquitin ligase MDM2 - that exerts its regulatory functions on p53 by poly-ubiquinating it for proteasomal degradation. Under normal conditions p53 and MDM2 are constituted in an interdependent negative-feedback loop that controls their levels. There are multiple examples in the literature that describe inhibition of MDM2 using small molecules that activate p53 and induce p53-mediated molecular processes, primarily in tumor cells. However, currently the range of potential applications of MDM2 inhibitors is not restricted to cancer, but also includes autoimmune diseases. Therefore, suppression of MDM2 by small molecules is considered as one of the major strategies for activation of p53 and its transcriptional functions, further leading to expression of p53-dependent genes.To evaluate the effect of small molecule p53-MDM2 inhibitors (Nutlin-3a and RG-7112) we have isolated peripheral blood mononuclear cells (PBMCs) of MS patients and healthy donors. First, using colorimetric MTS assay we determined cytotoxicity of the compounds in PBMCs for 0.001-100 uM concentration range and incubation time of 24, 48, 72 hours. It was found that both compounds have only minor effect on cell viability in tested conditions.Finally, we employed Taqman real-time reverse transcription PCR (RT-PCR) to quantitatively analyze gene expression with the use of fluorescence-based detection system BioRad CFX-96 Touch. Freshly isolated cells were treated with both compounds at 5-20 uM concentrantion range. Total RNA was isolated and used to synthesize cDNA via reverse transcription reaction and then real-time PCR was carried out. Relative normalized expression was calculated by normalization to beta-actin, data analyzed using CFX Manager software. All PCR reactions were performed in triplicates. The effect of PBMC treatment was investigated on expression levels of key p53 target genes such as p21, Mdm2, PUMA, GADD45A, BAX, Bcl2 . We observed concentration-dependent expression of p21, Mdm2, Puma, GADD45A and Bax genes. Interestingly, Nutlin-3a demonstrated a more profound effect on these genes compared to RG-7112. Neither Nutlin-3a nor RG-7112 displayed any noticeable effect on the expression of Bcl2 . Importantly, we also noticed that gradual increase in Nutlin-3a concentration leads to higher expression levels of p53-dependent genes in MS patient samples compared to healthy donor samples. In conclusion, we expect that specific MDM2 inhibitors such as Nutlin-3a and RG-7112 can be used for regulation of p53-mediated processes in MS.Further experiments are currently ongoing to examine the effect of p53 activation on expression of various cytokines in stimulated and non-stimulated cells from both MS patients and healthy donors.The study was funded by RFBR research grant 16-34-60213 mol_а_dk. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fluorescent Probes for Selective Recognition of Hypobromous Acid: Achievements and Future Perspectives.

Reactive oxygen species (ROS) have been implicated in numerous pathological processes and their homeostasis facilitates the dynamic balance of intracellular redox states. Among ROS, hypobromous acid (HOBr) has a high similarity to hypochlorous acid (HOCl) in both chemical and physical properties, whereas it has received relatively little attention. Meanwhile, selective recognition of endogenous HOBr suffers great challenges due to the fact that the concentration of this molecule is much lower than that of HOCl. Fluorescence-based detection systems have emerged as very important tools to monitor biomolecules in living cells and organisms owing to distinct advantages, particularly the temporal and spatial sampling for in vivo imaging applications. To date, the development of HOBr-specific fluorescent probes is still proceeding quite slowly, and the research related to this area has not been systematically summarized. In this review, we are the first to review the progress made so far in fluorescent probes for selective recognition and detection of HOBr. The molecular structures, sensing mechanisms, and their successful applications of these probes as bioimaging agents are discussed here in detail. Importantly, we hope this review will call for more attention to this rising field, and that this could stimulate new future achievements.

Non-Invasive Diagnostic System Based on Light for Detecting Early-Stage Oral Cancer and High-Risk Precancerous Lesions-Potential for Dentistry.

Simple SummaryThe early detection of oral cancer and oral potentially malignant disorders can facilitate minimum intervention and subsequent improvements in prognosis and quality of life after treatment. Recently, several non-invasive adjunctive fluorescence-based detection systems have improved the accuracy of detection and diagnosis of oral cancer and oral potentially malignant disorders. This article summarizes current knowledge about fluorescence-based diagnostic methods and discusses their benefits and drawbacks from a clinical viewpoint.Oral health promotion and examinations have contributed to the early detection of oral cancer and oral potentially malignant disorders, leading to the adaptation of minimally invasive therapies and subsequent improvements in the prognosis/maintenance of the quality of life after treatments. However, the accurate detection of early-stage oral cancer and oral epithelial dysplasia is particularly difficult for conventional oral examinations because these lesions sometimes resemble benign lesions or healthy oral mucosa tissues. Although oral biopsy has been considered the gold standard for accurate diagnosis, it is deemed invasive for patients. For this reason, most clinicians are looking forward to the development of non-invasive diagnostic technologies to detect and distinguish between cancerous and benign lesions. To date, several non-invasive adjunctive fluorescence-based detection systems have improved the accuracy of the detection and diagnosis of oral mucosal lesions. Autofluorescence-based systems can detect lesions as a loss of autofluorescence through irradiation with blue-violet lights. Photodynamic diagnosis using 5-aminolevulinic acid (ALA-PDD) shows the presence of very early oral cancers and oral epithelial dysplasia as a red fluorescent area. In this article, currently used fluorescence-based diagnostic methods are introduced and discussed from a clinical point of view.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Diagnodent and VistaCam may be unsuitable for the evaluation of dental caries in archeological teeth.

The aim of the study was to compare the usefulness of fluorescence-based caries detection systems (Diagnodent and VistaCam) for the assessment of carious lesions on archeological molars. The study material consisted of teeth from the Cemetery of St. Mary Magdalene (Cmentarz św. Marii Magdaleny) in Wrocław, Poland. A sample of 178 permanent molars from 38 skulls were examined. Five surfaces of teeth (occlusal, mesial, distal, buccal, and lingual) were assessed on either basically cleaned or sandblasted teeth. Six diagnostic methods were used to detect carious lesions: the visual classification of the International Caries Detection and Assessment System (ICDAS II), fluorescent methods (Diagnodent and VistaCam), X-ray, cone beam computed tomography and histological sections. The sensitivity and specificity of the methods were determined using receiver operating characteristic (ROC) curves and the correlation between the severity of dental caries and the readouts obtained with each method. In most cases, Diagnodent and VistaCam yielded unsatisfactory specificity and sensitivity values. The area under curve (AUC) values in ROC curves for Diagnodent and Vistacam were lower than the AUC values obtained for the ICDAS II visual classification. According to our results, in the case of archeological teeth, neither Diagnodent nor VistaCam can be regarded as a better diagnostic method than the ICDAS II visual classification of caries.

The synergistic enhancement of silver nanocubes and graphene oxide on surface plasmon-coupled emission

The enhancement of surface plasmon-coupled emission (SPCE) by the synergistic effect of silver nanocubes (AgNCs) and graphene oxide (GO) on gold film has been observed with the enhancement factor over 30. The enhancement mechanisms were investigated through simulating the electromagnetic (EM) field patterns of near field and testing different concentration of AgNCs and thickness of dye layer. The enhancement was mainly triggered by the high electromagnetic field of AgNCs, the interaction between localized surface plasmons (LSP) and propagating surface plasmons (PSP) and the assistance of GO. This synergistic enhancement strategy provides a simple way to increase SPCE signal and enable develop a new fluorescence-based detection system.

Portable fluorescence-based microRNA detection system based on isothermal signal amplification technology.

MicroRNAs (miRNAs) diagnostics can be useful for diagnosing or confirming miRNA abundance and are used in screening tests and to assess changes in miRNAs in vivo. At present, the use of traditional nucleic acid amplification assays to detect miRNAs has been limited in laboratory environment because of the time, equipment, and technical expertise required to perform these assays. A specialized, rapid affordable miRNA detection system is necessary when there are limited resources or point-of-care testing needs. We designed a portable and affordable fluorescence-based miRNA detection system based on isothermal signal amplification technology, using SYBR Green II as a fluorescent dye. To reduce costs, we chose LED as a light source and designed the corresponding optical path for LED. The portable detection system shows results consistent with those by real-time PCR, and can be used to detect miR-183 with a limit of detection of approximately 2fmol. We used the system to detect miR-183 in tissues and blood from patients with hepatocellular carcinoma (HCC). The results from the portable detection device were compared with those from clinical trials and indicated that the miR-183 fluorescence signal could successfully identify HCC and provide information related to cancer progression.

Cellular Models for the Study of Prions.

It is now established that numerous amyloid proteins associated with neurodegenerative diseases, including tau and α-synuclein, have essential characteristics of prions, including the ability to create transmissible cellular pathology in vivo. We have developed cellular bioassays that report on the various features of prion activity using genetic engineering and quantitative fluorescence-based detection systems. We have exploited these biosensors to measure the binding and uptake of tau seeds into cells in culture and to quantify seeding activity in brain samples. These cell models have also been used to propagate tau prion strains indefinitely in culture. In this review, we illustrate the utility of cellular biosensors to gain mechanistic insight into prion transmission and to study neurodegenerative diseases in a reductionist fashion.

Intratumoral gene expression of 5-fluorouracil pharmacokinetics-related enzymes in stage I and II non-small cell lung cancer patients treated with uracil-tegafur after surgery: A prospective multi-institutional study in Japan

ObjectivesThis investigation was conducted to assess the use of the intratumoral mRNA expression levels of nucleic acid-metabolizing enzymes as biomarkers of adjuvant chemotherapy for non-small cell lung cancer (NSCLC) using uracil-tegafur in a multi-institutional prospective study. Materials and methods236 patients with a completely resected NSCLC (adenocarcinoma and squamous cell carcinoma) of pathological stage IA (maximum tumor diameter of 2cm or greater), IB, and II tumors were given a dose of 250mg of uracil-tegafur per square meter of body surface area per day orally for two years after surgery. Intratumoral mRNA levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) genes relative to an internal standard, β-actin, were determined using laser-capture microdissection and fluorescence-based real time PCR detection systems. Results and conclusionAmong 5-FU target enzymes, TS was the only one that showed a significant difference in the level of gene expression between the high and low gene expression groups, for both disease-free survival (DFS) and overall survival (OS), when patients were divided according to median values; 5-year DFS rates in high/low TS gene expression were 60.4% and 72.6%, respectively (p=0.050), 5-year OS rates were 78.1% and 88.6%, respectively (p=0.011). Cox's proportional hazard model indicated that the pathological stage and TS gene expression level were independent values for predicting DFS.The TS gene expression level was shown to be an independent predictive factor for DFS in stage I and II NSCLC patients who were treated with uracil-tegafur following surgery.

Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993U/mg).

Rapid detection of viral RNA by a pocket-size real-time PCR system

We present an economical, battery-powered real-time polymerase chain reaction (RT-PCR) system suitable for field and point-of-care applications; it has a built-in thermal management, a fluorescence-based detection system, and a single chip controller with a graphic touch-screen display.

Magnetic bead-based detection of autoimmune responses using protein microarrays

In the present study, a magnetic bead-based detection approach for protein microarrays is described as an alternative approach to the commonly used fluorescence-based detection system. Using the bead-based detection approach with applied magnetic force, it was possible to perform the detection step more rapidly as a result of the accelerated binding between the captured analyte in the microspot and the detection antibody, which was coupled to the magnetic beads. The resulting strong opacity shift on the microspots could be recorded with an ordinary flatbed scanner. In the context of autoimmunity, a set of 24 serum samples was analyzed for the presence of antibodies against 12 autoantigens using standard fluorescence and magnetic bead-based detection methods. Dynamic range, sensitivity, and specificity were determined for both detection methods. We propose from our findings that the magnetic bead-based detection option provides a simplified and cost effective readout method for protein microarrays.

Label-Free DNA Detection on Nanostructured Ag Surfaces

The dramatic local electric-field enhancement property of Ag nanoparticles was used as the basis to significantly increase the signal output of a novel label-free (or "self-labeled") fluorescence-based DNA detection system. In response to identical amounts of analyte, nanostructured Ag substrates provided a posthybridization fluorescent sensor response over 10-fold larger than the response from planar Au substrates. Detection performance strongly depended upon the Ag substrate roughness. Consistent with work by others on metal-enhanced fluorescence, fluorescence intensity also depended strongly on the distance between the fluorophore and the Ag substrate surface. Adjusting the surface roughness, amount of the Ag deposited on the surface, and the DNA probe length allowed for production of an optimized response. In addition to constituting a novel label-free DNA sensor, we anticipate that these structures will provide a unique platform for testing concepts in plasmonics.

Apo[a] size and PNR explain African American-Caucasian differences in allele-specific apo[a] levels for small but not large apo[a

Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size, we evaluated the upstream C/T and pentanucleotide repeat (PNR) polymorphisms. We determined apo[a] sizes, allele-specific apo[a] levels, and C/T and PNR in 215 Caucasians and 139 African Americans. For Caucasians, apo[a] size affected allele-specific levels substantially greater in subjects with apo[a] < 24 K4; for African Americans, the size effect was smaller than in Caucasians, <24 K4, but did not decrease at higher repeats. In both groups, the level decreased with increasing size of the other allele. Controlling for apo[a] sizes, PNR decreased allele-specific apo[a] levels in Caucasians with increasing PNR > 8. In a multiple regression model, apo[a] allele size and size and expression of the other apo[a] allele (and PNR > 8 for Caucasians) significantly predicted allele-specific apo[a] levels. For a common PNR 8 allele, predicted values were similar in the two ethnicities for small size apo[a]. Allele-specific apo[a] levels were influenced by the other allele size and expression. Observed differences between Caucasians and African Americans in allele-specific apo[a] levels were explained for small apo[a] sizes by the other allele size and PNR; the ethnicity differences remain unexplained for larger sizes.

Cross-Talk Problem on a Fluorescence Multi-Channel Microfluidic Chip System

Development of a compact fluorescence-based detection system for use in a micro-analytical system, such as a point-of-care diagnostic system, often requires a multi-channel microfluidic chip system. Since the materials used for microfluidic chips usually are transparent in the visible region and have a refractive indices higher than that of air or the surrounding environment, the fluorescence emission and scattered excitation light can propagate through the chip. We observed that such propagation can cause cross-talk between adjacent channels, and may become the major source of noise in the system and/or photo bleach the fluorescent samples in the adjacent channels, particularly for the small distances between the channels found in microfluidic chips, usually in order of several micro m. We monitored this cross-talk using fluorescein as a fluorescent sample and Mylar sheeting as a microfluidic chip material. We then discuss how this cross-talk can be avoided using a simple, inexpensive and effective method.

Diminished lipoxin biosynthesis in severe asthma.

Severe asthma is characterized by increased airway inflammation that persists despite therapy with corticosteroids. It is not, however, merely an exaggeration of the eosinophilic inflammation that characterizes mild to moderate asthma; rather, severe asthma presents unique features. Although arachidonic acid metabolism is well appreciated to regulate airway inflammation and reactivity, alterations in the biosynthetic capacity for both pro- and antiinflammatory eicosanoids in severe asthma have not been determined. Patients with severe asthma were identified according to National Heart, Lung, and Blood Institute Severe Asthma Research Program criteria. Samples of whole blood from individuals with severe or moderate asthma were assayed for biosynthesis of lipoxygenase-derived eicosanoids. The counterregulatory mediator lipoxin A4 was detectable in low picogram amounts, using a novel fluorescence-based detection system. In activated whole blood, mean lipoxin A4 levels were decreased in severe compared with moderate asthma (0.4 [SD 0.4] ng/ml vs. 1.8 [SD 0.8] ng/ml, p=0.001). In sharp contrast, mean levels of prophlogistic cysteinyl leukotrienes were increased in samples from severe compared with moderate asthma (112.5 [SD 53.7] pg/ml vs. 64.4 [SD 24.8] pg/ml, p=0.03). Basal circulating levels of lipoxin A4 were also decreased in severe relative to moderate asthma. The marked imbalance in lipoxygenase-derived eicosanoid biosynthesis correlated with the degree of airflow obstruction. Mechanisms underlying airway responses in severe asthma include underproduction of lipoxins. This is the first report of a defect in lipoxin biosynthesis in severe asthma, and suggests an alternative therapeutic strategy that emphasizes natural counterregulatory pathways in the airways.