Fluorescence Assay System Research Articles

Multi-aptamer-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of S. pneumoniae and S. aureus.

A novel nucleic acid aptamer nanoprobes-mediated hairpin allosteric and aptamer-assisted CRISPR system for detection of Streptococcus pneumoniae and Staphylococcus aureus is presented.In this fluorescence assay system, utilizing the hairpin allosteric effect caused by the aptamer binding to the target bacteria, the detection of S. pneumoniae is first achieved through changes in fluorescence due to FRET. Subsequently, aCas12a protein mixture is added to detect S. aureus. The amplified output signal is triggered by two methods to ensure the sensitivity of the method: the synergistic FRET effect is achieved by the assembly of multi-aptamer through the conjugation of streptavidin-biotin, and the trans-cleavage function of CRISPR/Cas 12a. Under the optimized conditions, the proposed hairpin allosteric aptasensor could achieve high sensitivity (a detection limit of 135cfu/mL) and broad-concentration quantification (dynamic range of 103-107cfu/mL) of S. pneumoniae. The aptamer-assisted CRISPR system for S. aureus detection showed good linearity (R2 = 0.996) in the concentration range 102-108cfu/mL, with a detection limit of 39cfu/mL. No cross-reactivity with other foodborne pathogenic bacteria was observed in both systems. Taking only 55min, this method of multiple pathogen detection proved to be promising.

Application of Enzyme-Linked Fluorescence Assay (ELFA) to Obtain In Vivo Matured Dog Oocytes through the Assessment of Progesterone Level.

Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D - 6 to D + 1) in both systems were compared. The ELFA range of 5.0-12.0 ng/mL was derived from the RIA standard range of 4.0-8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.

Three distinct mechanisms underlying human γδ T cell-mediated cytotoxicity against malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive thoracic tumor with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors exhibit a promising effect in some patients with unresectable MPM in clinical trials, the majority of MPM patients show only modest response rates to the currently available treatments. It is thus imperative to develop novel and innovative therapeutic modalities for MPM, including immune effector cell-based therapies. γδ T cells were expanded using tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) and interleukin-2, and the therapeutic potential of γδ T cells was examined through analyzing cell surface markers and cellular cytotoxicity against MPM in vitro using a europium chelate-based time-resolved fluorescence assay system and a luciferase-based luminescence assay system. We successfully expanded γδ T cells from peripheral blood mononuclear cells of healthy donors and MPM patients. γδ T cells expressed natural killer receptors such as NKG2D and DNAM-1 and exhibited a moderate level of cytotoxicity to MPM cells in the absence of antigens. The inclusion of PTA, (E)-4-hydroxy-3- methylbut-2-enyl diphosphate (HMBPP) or zoledronic acid (ZOL) induced a TCR-dependent cytotoxicity in γδ T cells and secreted interferon-γ (IFN-γ). In addition, γδ T cells expressing CD16 exhibited a significant level of cytotoxicity against MPM cells in the presence of an anti-epidermal growth factor receptor (EGFR) mAb, at lower concentrations than in clinical settings, whereas a detectable level of IFN-γ was not produced. Taken together, γδ T cells showed cytotoxic activity against MPM in three distinct mechanisms through NK receptors, TCRs and CD16. Since major histocompatibility complex (MHC) molecules are not involved in the recognition, both autologous and allogeneic γδ T cells could be used for the development of γδ T cell-based adoptive immunotherapy for MPM.

A dual-response fluorescent probe for detection and bioimaging of hydrazine and cyanide with different fluorescence signals

Due to favourable efficiency and low cost, dual-response fluorescent probes play critical roles in the development of fluorescence assay system. Herein, a novel dual-response fluorescent probe (RDCN) was designed and synthesized for the detection of two environmental contaminants: hydrazine (N2H4) and cyanide (CN−). Probe RDCN exhibited discriminative sensing behaviors to N2H4 and CN− with different reaction mechanisms, allowing the high selectivity and sensitivity detection for N2H4 and CN−. The probe itself displayed red-emitting fluorescence as a result of strong intramolecular charge transfer (ICT) between diethylamino and dicyano. After mixing with hydrazine, a new corresponding hydrazone came into being with an intense yellow fluorescence. While, the probe could also largely switch to blue fluorescence in response to CN−. Furthermore, the probe RDCN was successfully employed to determine N2H4 and CN− in water samples with the detection limits of 0.08 μM and 0.33 μM, and to visualize N2H4 and CN− in live cells by means of different fluorescence channels (blue and yellow channels), revealing that probe RDCN has potential applications for discriminative detection N2H4 and CN− in biological environment.

Preliminary verification studies of the motility and fluorescence assay (MFA) for ballast water quality monitoring

The International Maritime Organization (IMO) has adopted a ballast water convention to prevent spread of invasive species with ballast water and with it comes the need for fast and reliable tests to estimate the number of viable organisms found in ballast water.During 2017 and 2018, computer-assisted automatic quantification of plankton was compared with direct microscopic counts. Analysed samples represented simulations of ballast water samples from Denmark. Samples consisted of live plankton monocultures, mixed cultures, or ambient organisms from local seawater. The aim was to validate the performance and efficiency of the automated Motility and Fluorescence Assay (MFA) system BallastWISE, which automatically counts motile and fluorescent organisms in the fractions ≥10 - <50 μm and ≥ 50 μm, according to the ballast water discharge standard.Standardized counting methods for comparison were completed under microscope. Organisms in the ≥10 - <50 μm size range were analysed using an FDA (Fluorescein diacetate) and CMFDA (5-chloromethylfluorescein diacetate) staining method, and a standard movement and response to stimuli technique was used for the ≥50 μm size range.BallastWISE results correlated well with direct microscopic counts, showing promising counting performance and efficiency when it comes to rapid analysis of ballast water plankton samples. Quantification of monoculture and mixed cultures using BallastWISE correlated well with manual viable counts (Tetraselmis suecica in size range ≥ 10 - <50 μm: Pearson correlation (r) = 1.0; Tigriopus californicus and Brachionus plicatilis in size range ≥ 50 μm: r = 0.99). BallastWISE counts also correlated well with viable microscopic counts of ambient organisms in natural seawater (size range ≥ 10 - <50 μm: r = 0.97 and size range ≥ 50 μm: r = 0.89). We propose that computer-assisted quantification of plankton provides a rapid and precise alternative to traditional counting and identification of organisms in water samples such as ballast water.

Optical measurement of glutamate in slice preparations of the mouse retina

Signaling by glutamatergic synapses plays an important role in visual processing in the retina. In this study, we used an enzyme-linked fluorescence assay system to monitor the dynamics of extracellular glutamate in a slice preparation from the mouse retina. High K stimulation induced an elevation of fluorescence in the inner plexiform layer (IPL) of the retina when glutamate transporters were inhibited by dl-threo-β-benzyloxyaspartic acid (TBOA). The high K-induced fluorescence signals in the IPL were inhibited by the calcium channel blocker Cd2+. Blockade of GABAergic and glycinergic circuits by picrotoxin and strychnine also elevated the fluorescence signals in the IPL. Thus, the enzyme-linked fluorescence assay system might be useful for monitoring the bulk concentration of extracellular glutamate released by synapses in the inner retina.

Facile one-step synthesis of highly luminescent N-doped carbon dots as an efficient fluorescent probe for chromium(vi) detection based on the inner filter effect

Label-free fluorescence assay system is designed for Cr(iv) detection.

Quantitative analysis by flow cytometry of green fluorescent protein-tagged human phenylalanine hydroxylase expressed in Dictyostelium

Abstract We have developed a fluorescence assay system to monitor the protein levels of human phenylalanine hydroxylase (hPAH). Wild-type (WT) and three mutant hPAHs (I65T, L255V, and S349L) were expressed as green fluorescent protein (GFP)-tagged forms in a PAH knockout mutant (pah −) of Dictyostelium discoideum Ax2. The fluorescence-activated cell sorting (FACS) analysis showed that the GFP positive cells were the most frequent in WT but were rare in pah −, demonstrating the successful expression of GFP-tagged hPAHs in Dictyostelium. The fluorescence levels of mutants relative to WT were higher than expected from the protein amounts determined from the non-tagged forms, probably due to the presence of the N-terminal GFP. However, treatment of the cells with cumene hydroperoxide, which is known to accelerate protein degradation, decreased fluorescence levels, suggesting that protein stability changes in individual mutations can be monitored by FACS analysis. For an evaluation study, a putative pharmacological chaperone effect of yeast extract on S349L was examined by Western blot and FACS analysis. Both the protein amount and the fluorescence levels were increased by yeast extract, supporting that the FACS analysis could replace the time- and labor-consuming procedures such as the Western blot and cell culture. The fluorescence-based cell assay system may be valuable for the high-throughput screening of pharmacological chaperones for phenylketonuria mutations.

New artificial fluoro-cofactor of hydride transfer with novel fluorescence assay for redox biocatalysis.

A new artificial fluoro-cofactor was developed for the replacement of natural cofactors NAD(P), exhibiting a high hydride transfer ability. More importantly, we established a new and fast screening method for the evaluation of the properties of artificial cofactors based on the fluorescence assay and visible color change.

막과산화를 신속히 유발하는 제초제의 고효율 대량스크리닝을 위한 형광검정법

This study was conducted to establish a fluorescence assay system for high efficient mass screening of the herbicides causing rapid membrane peroxidation, based on the fact that peroxide in cellular leakage could be fluorometrically determined through the fuorescent compounds formed after reacting with homovanillic acid (HVA) and peroxidase (HRP). The assay procesure established in this study was as follows. Only single disc (4 mm diameter) excised from cucumber cotyledon is placed on the well containing test solution (200 μL) with 96-well microplate. The plate is shaking-incubated for 8 h under light condition. Then after removing the cucumber disc, HVA and HRP are supplied in the medium buffer and incubated for 5 min at room temperature. Fluorescence values are determined at Ex 320 nm/Ex 425 nm. The higher fluorescence values are obtained in the treatment of chemical having higher herbicidal activity. Using this assay with 96-well microplates, a large number of herbicides inducing rapid membrane peroxidation seemed to be screened more efficiently than spectrophotometric microtiter assay reported previously.

Fluorescence Immunoassay of Human D‐dimer in Whole Blood

D-dimer is a widely used biomarker for the initial clinical assessment of suspected deep vein thrombosis and pulmonary embolism. Here, we presented a new fluorescence (FL) D-dimer assay system, which was developed with a platform of point-of-care test (POCT) for clinical applications. Whole blood was mixed with FL-labeled anti-D-dimer detector antibody, and then loaded onto a disposable cartridge. After 12 min of incubation, the FL intensity was acquired by scanning of test cartridge and converted as level of D-dimer in a laser FL scanner. The analytical performance of FL immunoassay was evaluated by linearity, recovery, and precision tests. The comparability of the developed assay was examined with automated reference methods. The FL assay system showed a good linearity, and the analytical mean recovery of control was 103% in a dynamic working range. The imprecision of intra- and inter-as-say of coefficient of variations from assay system was less than 8%. The developed FL assay system showed strong correlation with two automated reference assays, Vidas D-dimer (r = 0.973) and Stalia D-dimer (r = 0.971). The new FL immunoassay for D-dimer is a user-friendly, precise, and reproducible platform of POCT in whole blood.

Fluorogenic aldehydes bearing arylethynyl groups: turn-on aldol reaction sensors for evaluation of organocatalysis in DMSO

Fluorogenic aromatic aldehydes bearing arylethynyl groups were developed. They were used for monitoring the reaction progress of organocatalytic aldol reactions in DMSO through an increase in the fluorescence intensity based on the formation of the florescent aldol product. The ratios of the fluorescence intensities of the aldols to the aldehydes were more than 300. These results suggest that the fluorescence assay system using the aldehyde is useful for the rapid identification of superior aldol catalysts and reaction conditions.

1P312 Yeast-based fluorescence assay system for detecting human G protein-coupled receptor activation in water-in-oil droplets(28. Bioengineering,Poster)

1P312 Yeast-based fluorescence assay system for detecting human G protein-coupled receptor activation in water-in-oil droplets(28. Bioengineering,Poster)

H2O2‐ and pH‐sensitive CdTe quantum dots as fluorescence probes for the detection of glucose

A novel fluorescence assay system for glucose was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as probes. The luminescence quantum yield of the TGA-capped CdTe QDs was highly sensitive to H2 O2 and pH. In the presence of glucose oxidase, glucose is oxidized to yield, gluconic acid and H2 O2 . H2 O2 and H(+) (dissociated from gluconic acid) intensively quenched the fluorescence of QDs. The experimental results showed that the quenched fluorescence was proportional to the glucose concentration within the range of 0.01-5.0 mm under optimized experimental conditions. Compared with most of the existing methods, this newly developed system possesses many advantages, including simplicity, low cost, high flexibility, and good sensitivity. Furthermore, no complicated chemical modification of QDs and enzyme immobilization was needed in this system.

Characterization of P2X7Purinergic Receptors and Their Function in Rat Lacrimal Gland

To characterize the effects of P2X(7) purinergic receptors on lacrimal gland function. P2X(7) purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Rat lacrimal gland acini were isolated by collagenase digestion. Acini were incubated with the fluorescent indicator molecule fura 2, and [Ca(2+)](i) was measured by a fluorescence imaging system. Protein secretion was measured with a fluorescence assay system. Activation of ERK 1/2 was determined by Western blot analysis with an antibody against phosphorylated (active) ERK 1/2. P2X(7) receptors were present in the lacrimal gland by RT-PCR and Western blot analysis. These receptors were located in the membranes of acinar and ductal cells and the cytoplasm of acinar cells. Activation of P2X(7) receptors with (benzoylbenzoyl)adenosine 5'-triphosphate increased [Ca(2+)](i), peroxidase secretion, and ERK 1/2 activation, each of which was inhibited by the P2X(7) receptor inhibitors Brilliant Blue G or A 438079. P2X(7) purinergic receptors are present in rat lacrimal gland and when stimulated increase [Ca(2+)](i), protein secretion, and ERK 1/2 activation.

Real-time fluorescence monitoring of GSK3β-catalyzed phosphoryation by use of a BODIPY-based Zn(II)–Dpa chemosensor

We developed a new fluorescence assay system for GSK3β-catalyzed kinase reaction using the BODIPY-based fluorescent chemosensor. This system exploits the selective sensing property of the chemosensor for a (i, i+4) bis-phosphorylated peptide, which allows us to conveniently detect the phosphorylation reaction with a fluorescence increase in a real-time fashion.

Convenient and efficient synthesis of a lanthanide3+‐coordinated, diethylene triamine pentaacetic acid labeled biopolymer as an assay for the cholecystokinin B receptor

To develop an assay for the cholecystokinin B receptor with an Eu(3+)-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide(3+)-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study.

A Real-Time Fluorescence Assay for MeasuringN-Dealkylation

A real-time fluorescence assay system using a series of 9-N-(alkylamino)acridine derivatives (methyl, ethyl, n-propyl, n-butyl, n-pentyl, and benzyl) that are N-dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is approximately 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYP1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N-dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N-dealkylated methyl, ethyl, n-propyl, and n-butyl substrates, whereas CYP1A1 N-dealkylated these plus the n-pentyl derivative. Activities using 5 microM 9-N-(alkylamino)acridine substrates ranged from 0.1 to 0.9 pmol 9AA/min/pmol P450. Kinetic constants for CYP1A1 N-dealkylation of the 9-N-(methylamino)acridine (MAA) and 9-N-(ethylamino)acridine (EAA) were K(m) 1.09 +/- 0.68 and 0.35 +/- 0.21 microM and the V(max) 61.9 +/- 48.5 and 113.8 +/- 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N-dealkylation of MAA and EAA were K(m) 7.9 +/- 5.4 and 3.2 +/- 1.6 microM, and V(max) 501 +/- 35.4 and 702.7 +/- 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (DeltaG(bind)) were calculated for MAA with CYP1A1 and CYP2D6 to be -8.266 and -7.074 kcal/mol, respectively. The DeltaG(bind) values for EAA with CYP1A1 and CYP2D6 were -8.950 and -7.618 kcal/mol, respectively. The substrates were suitable for monitoring N-dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.

Nitric Oxide and cGMP Mediate α1D-Adrenergic Receptor–Stimulated Protein Secretion and p42/p44 MAPK Activation in Rat Lacrimal Gland

To determine whether alpha(1)-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland. Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an alpha(1)-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10(-4) M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK. eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N(G)-nitro-l-arginine methyl ester (l-NAME; 10(-6) M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the alpha(1D)-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation. alpha(1D)-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.

Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol beta-aryl ether linkages.

Cleavage of the arylglycerol beta-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial beta-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the beta-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The beta-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The beta-aryl ether cleavage enzyme converts the guaiacylglycerol beta-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the C alpha alcohol structure and p-hydroxyl group and specifically attacks the beta-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the C alpha and C beta positions.