Sensitive fluorescent detection of protein on nylon membranes

Detection of antigen immobilized on membranes, as in Western transfers and dot enzyme linked immunosorbent assays (ELISAs), often employ antibody-enzyme conjugates and chemiluminescent or precipitated colored reaction products. Although chemiluminescent markers are sensitive, they are time-consuming because of their required exposure to X-ray film and the presence of background artifacts sometimes limits their use. This report demonstrates that direct fluorescent detection technique using nylon membranes that has higher sensitivity than chemiluminescent methods is easier to perform and has a uniform, low background. An alkaline phosphatase conjugated antibody was compared with antibody conjugated to a fluorescent phycobiliprotein (allophycocyanin) for sensitivity in both Western transfers and dot ELISA assays using mouse IgG as the membrane-bound antigen. Direct fluorescent detection of antigen-antibody complexes on positively charged nylon membrane provided better sensitivity and lower background than similar conditions using enzyme amplification and chemiluminescent detection on either nylon or PVDF membranes. Processing time was reduced by the elimination of steps associated with substrate incubation, washing and X-ray film exposures required for chemiluminescence detection. These data support the view that direct fluorescent detection can represent a significant improvement in assay sensitivity and reduction in time compared with more traditional chemiluminescent detection techniques employed in the conduct of Western transfers and dot ELISA studies.     

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.     

A green fluorescent protein-based whole-cell bioreporter for the detection of phenylacetic acid

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAACoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.     

Sensitive detection of cysteine based on fluorescent silver clusters

In this work, we report the application of novel, water-soluble fluorescent Ag clusters in fluorescent sensors for detecting cysteine, an important biological analyte. The fluorescence of poly(methacrylic acid) (PMAA)-templated Ag clusters was found to be quenched effectively by cysteine, but not when the other alpha-amino acids were present. By virtue of the specific response, a new, simple, and sensitive fluorescent method for detecting cysteine has been developed based on Ag clusters. The present assay allows for the selective determination of cysteine in the range of 2.5 x 10(-8) to 6.0 x 10(-6)M with a detection limit of 20 nM at a signal-to-noise ratio of 3. Based on the absorption and fluorescence studies, we suggested that cysteine quenched the emission by the thiol-adsorption-accelerated oxidation of the emissive Ag clusters. The present study shows a promising step toward the application of silver clusters, a new class of attractive fluorescence probes.     

Improving membrane voltage measurements using FRET with new fluorescent proteins

We used two new coral fluorescent proteins as fluorescence resonance energy transfer (FRET) donor and acceptor to develop a voltage sensor, named Mermaid, that displays approximately 40% changes in emission ratio per 100 mV, allowing for direct visualization of electrical activities in cultured excitable cells. Notably, Mermaid has fast on-off kinetics at warm (approximately 33 degrees C) temperatures and can report voltage spikes comparable to action potentials.     

Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers.

Enteropathogenic YERSINIA: bacteria trigger the production of the proinflammatory chemokine IL-8, an important chemokine for the recruitment of polymorphonuclear leukocytes (PMN). YERSINIA: is resistant to phagocytosis by PMN, and the recruitment of these cells is thought to be part of a pathogenic strategy of YERSINIA: to establish infection by allowing the pathogen to gain access to, and disseminate within, host tissue. We report here that YERSINIA: expressing the outer membrane protein invasin triggers IL-8 production in epithelial cells. The 195 carboxyl-terminal amino acids of invasin when linked to latex beads are sufficient to trigger IL-8 production. By means of IL-8 promoter reporter gene assays and electrophoretic mobility shift assay experiments, the minimal optimal region of the IL-8 promoter responsive to invasin was identified and invasin-responsive control elements were characterized. Invasin-induced activation of the IL-8 promoter was found to be mediated through a previously identified NF-kappaB element. This NF-kappaB binding site preferentially binds Rel p65-p65 homodimers as well as some p50-p65 heterodimers in response to stimulation by invasin. Invasin-induced NF-kappaB activation correlated with degradation of IkappaBalpha and the inhibition of NF-kappaB by specific inhibitors of IkappaB activation blocked invasin-induced IL-8 secretion. Invasin-triggered IL-8 production does not depend on invasin-triggered uptake of bacteria, and is independent of a functional PI3-kinase. This report is the first to demonstrate the molecular basis of IL-8 production triggered by enteropathogenic bacteria. Together, these data elucidate the possible early pathomechanisms operating in YERSINIA: infection and may have implications for the design of novel therapeutics directed against this enteropathogen.     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Myotrophin/V-1, a protein up-regulated in the failing human heart and in postnatal cerebellum, converts NFkappa B p50-p65 heterodimers to p50-p50 and p65-p65 homodimers

Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of kappaB (NFkappaB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFkappaB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IkappaBalpha without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFkappaB proteins in vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFkappaB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adbetagal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFkappaB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced kappaB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFkappaB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFkappaB dimers during this period.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.     

Sensitive detection of p53 tumor suppressor gene using an enzyme-based solid-state electrochemiluminescence sensing platform

An enzyme-based solid-state electrochemiluminescence (ECL) sensing platform for sensitive detection of a single point mutation is developed successfully using p53 tumor suppressor gene as a model analyte. A composite of multiwalled carbon nanotubes and Ruthenium (II) tris-(bipyridine) (MWNTs-Ru(bpy)(3)(2+)) was prepared and coated on an electrode surface, which was covered by polypyrrole (PPy) to immobilize ssDNA. Then, the ssDNA recognized the gold nanoparticle (AuNP)-labeled p53 tumor suppressor gene, and produced AuNP-dsDNA electrode with AuNP layer. The surface adsorbed the glucose-dehydrogenase (GDH) molecules for producing ECL signal. This system combined enzyme reaction with ECL detection, and it can recognize sequence-specific wild type p53 sequence (wtp53) and muted type p53 sequence (mtp53) with discrimination of up to 56.3%. The analytic results were sensitive and specific. It holds promise for the diagnosis and management of cancer.     

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

Single-mismatch detection using gold-quenched fluorescent oligonucleotides

Here we describe a hybrid material composed of a single-stranded DNA (ssDNA) molecule, a 1.4 nm diameter gold nanoparticle, and a fluorophore that is highly quenched by the nanoparticle through a distance-dependent process. The fluorescence of this hybrid molecule increases by a factor of as much as several thousand as it binds to a complementary ssDNA. We show that this composite molecule is a different type of molecular beacon with a sensitivity enhanced up to 100-fold. In competitive hybridization assays, the ability to detect single mismatch is eightfold greater with this probe than with other molecular beacons.     

Evolutionary optimization of fluorescent proteins for intracellular FRET

Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.     

Green fluorescent protein-based system for analysis of E-selectin-mediated adhesion

Numerous cell-based or cell-free systems for study of selectin adhesion use radiolabeled tracers. However, in addition to handling problems associated with the use of radioisotopes, these assays have difficulty relating a number of counts to a number of adherent cells. Here, we describe an assay that uses the natural fluorescence of the green fluorescent protein (GFP) to measure binding of cells to E-selectin. We elaborated an adhesion system composed of a cell monolayer expressing E-selectin ligand to which monodispersed fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are added. Due to GFP autofluorescence, adhered cells can be easily distinguished from cell monolayers by fluorescence microscopy, and adhesion can be measured by cytofluorometry. We applied this GFP-based adhesion assay to measure the adherence of a pancreatic tumor cell line and found that the binding parameters of these cells satisfy a number of E-selectin-specific criteria.     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.