Generation Of Fluorescent Signal Research Articles

Sensing of biomolecules and label-free discrimination of DNA containing a triple T–C/T–G mismatch pair with a fluorescence light-up probe, triazolylpyrene (TNDMBPy)

Binding to the minor groove of calf-thymus DNA (ct-DNA) and strong binding in hydrophobic pocket of bovine serum albumin (BSA), switched-on the fluorescence of smart fluorescent probe, triazolylpyrene (TNDMBPy). Also, a novel label-free strategy was adopted to detect DNA base mismatch via the generation of distinct fluorescence signal at visible region utilizing the same probe

Distribution and accumulation of Cy5.5-labeled thermally cross-linked superparamagnetic iron oxide nanoparticles in the tissues of ICR mice

Free Cy5.5 dye and Cy5.5-labeled thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) have been routinely used for in vivo optical imaging. However, there is little information about the distribution and accumulation of free Cy5.5 dye and Cy5.5-labeled TCL-SPION in the tissues of mice. Free Cy5.5 dye (0.1 mg/kg body weight) and Cy5.5-labeled TCL-SPION (15 mg/kg body weight) were intravenously injected into the tail vein of ICR mice. The biodistribution and accumulation of the TCL-SPION and Cy5.5 were observed by ex vivo optical imaging and fluorescence signal generation at various time points over 28 days. Cy5.5 dye fluorescence in various organs was rapidly eliminated from 0.5 to 24 h post-injection. Fluorescence intensity of Cy5.5 dye in the liver, lung, kidney, and stomach was fairly strong at the early time points within 1 day post-injection. Cy5.5-labeled TCL-SPION had the highest fluorescence density in the lung at 0.5 h post-injection and decreased rapidly over time. Fluorescence density in liver and spleen was maintained over 28 days. These results suggest that TCL-SPION can be useful as a carrier of therapeutic reagents to treat diseases by persisting for long periods of time in the body.

Cell:cell interactions in the immune system

Video podcast available Go to www.immunologicalreviews.com to watch an interview with Guest Editor Facundo Batista. The cells of the immune system are continually on the move! Naive T cells in a human collectively crawl over 10 000 miles a day in search of antigens (1). Each dendritic cell (DC) touches 5000 T cells an hour to scan repertoires over a million deep (2, 3). The adaptive immune system functions through a vast network of provisional cell–cell interactions that are continuously changing. When this frenetic scanning finds a fit between T-cell antigen receptor (TCR) and DC antigen, the interaction can be prolonged with a pronounced deceleration of the T cell and finally a long-lived interaction that has been described as an immunological synapse (2, 4). In vitro studies on the immunological synapse suggest a profound supramolecular organization that serves focused trans-synaptic and juxtracrine information transfer between cells (5–8). Immunological synapses are not restricted to T cells but are also formed by natural killer (NK) cells, B cells, granulocytes, and phagocytes using related immunotyrosine-based activation motif-containing receptors (9, 10). These interactions are profoundly influenced by the three-dimensional (3D) tissue environment (11). This volume of Immunological Reviews takes a broader look at the cell–cell interactions in the immune system from the molecular organization of antigen receptors during surveillance to the organization of stromal cell networks in lymph nodes, bone marrow, and the lung (Fig. 1). The organization of the plasma membrane is a fundamental issue in biology that sets the stage for cell–cell interaction and communication. The classical fluid mosaic model suggested membrane proteins diffusing in a lipid sea with potential for interactions with cytoplasmic and extra-cytoplasmic binding partners that account for regulation and processes like adhesion and receptor capping. Observations on lipid phases in model systems with disordered, liquidordered, and gel phases in order of increasing viscosity suggested that such phases could also exist in biological membranes on some scale (12). Biophysical observations of liquid-ordered domains enriched in various lipid-modified proteins have led to models in which lipids have a role in providing a higher level of organization (13). Most recently, evidence for protein-driven domains, referred to as Immunological Reviews 2013 Vol. 251: 7–12 Printed in Singapore. All rights reserved

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label free nuclease enzymedetection

The double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) with high fluorescence, whereas the single-strand DNA (ssDNA) cannot support the formation of Cu NPs. This difference in fluorescent signal generation can be used for the detection of nuclease cleavage activity. Thus, a label-free strategy for sensitive detection of nuclease has been developed. The sensor contains a complete complementary dsDNA which acts as a template for the formation of Cu NPs and generation of fluorescence signal. The enzyme S1 nuclease was taken as the model analyte. Upon addition of S1 nuclease into the sensing system, the DNA was cleaved into fragments, preventing the formation of the Cu NPs and resulting in low fluorescence. In order to achieve the system's best sensing performance, a series of experimental conditions were optimized. Under the optimized experimental conditions, the sensor exhibits excellent performance (e.g., a detection limit of 0.3UmL−1 with high selectivity). This possibly makes it an attractive platform for the detection of S1 nuclease and other biomolecules.

A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter

In this paper, we reported a convenient fluorescence method for the detection of genetically modified organisms (GMOs). As it is known that the cauliflower mosaic virus (CaMV) 35S promoter is widely used in most transgenic plants (Schnurr and Guerra, 2000), we thus design a simple method based on the detection of a section target DNA (DNA-T) from the transgene CaMV 35S promoter. In this method, the full-length guanine-rich single-strand sequences were split into fragments (Probe 1 and 2) and each part of the fragment possesses two GGG repeats. In the presence of K+ ion and berberine, if a complementary target DNA of the CaMV 35S promoter was introduced to hybridize with Probe 1 and 2, a G-quadruplex–berberine complex was thus formed and generated a strong fluorescence signal. The generation of fluorescence signal indicates the presence of CaMV 35S promoter. This method is able to identify and quantify Genetically Modified Organisms (GMOs), and it shows wide linear ranges from 5.0×10−9 to 9.0×10−7mol/L with a detection limit of 2.0×10−9mol/L.

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37°C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R2 values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.

Characterization of sensitivity and specificity in leaky droplet-based assays

This paper uses numerical methods to characterize the crosstalk of small fluorescent molecules and molecular probes among aqueous droplets immersed in a continuous phase of hydrocarbons or fluorocarbons in microfluidic systems. Droplet-based biochemical assays rely on the reagents to remain isolated in individual droplets. It has been observed, however, that small and hydrophobic fluorescent molecules can diffuse across the droplet boundary into other drops. The contents among droplets become mixed and homogenized over time. Such cross-contamination can have detrimental effects on the accuracy of droplet-based assays, especially those using fluorescent molecules and the corresponding number of fluorescent droplets for a quantitative readout. This work examines the competing dynamics of the generation of fluorescent molecules in "positive" drops (in response to the presence of molecules or cells of interest), against its leakage into "negative" drops, where such molecules or cells of interest are absent. In ideal droplet assays, the signal-to-noise ratio (SNR)--defined as the fluorescence signal from a positive drop to that from a negative drop--would increase and saturate with time. In a leaky droplet assay, the SNR tends to decay with time. Under certain conditions, however, the SNR from a leaky droplet assay could increase and reach a maximum value before it starts to diminish. This maximum value can be estimated from a dimensionless number relating the rate of leakage relative to the rate of generation of fluorescence signal in the drops. Beyond the time when the SNR peaks, the SNR value, as well as the accuracy of the leaky droplet assay continues to degrade. In the absence of immediate experimental remedies to completely eliminate the crosstalk of molecules among drops, performing detection at the optimal time point becomes critical to minimize errors in leaky droplet assays.

Tumor-homing photosensitizer-conjugated glycol chitosan nanoparticles for synchronous photodynamic imaging and therapy based on cellular on/off system

Herein, we developed the photosensitizer, protoporphyrin IX (PpIX), conjugated glycol chitosan (GC) nanoparticles (PpIX–GC–NPs) as tumor-homing drug carriers with cellular on/off system for photodynamic imaging and therapy, simultaneously. In order to prepare PpIX–GC–NPs, hydrophobic PpIXs were chemically conjugated to GC polymer and the amphiphilic PpIX–GC conjugates formed a stable nanoparticle structure in aqueous condition, wherein conjugated PpIX molecules formed hydrophobic inner-cores and they were covered by the hydrophilic GC polymer shell. Based on the nanoparticle structure, PpIX–GC–NPs showed the self-quenching effect that is ‘off’ state with no fluorescence signal and phototoxicity with light exposure. It is due to the compact crystallized PpIX molecules in the nanoparticles as confirmed by dynamic light scattering and X-ray diffraction methods. However, after cellular uptake, compact nanoparticle structure gradually decreased to generate strong fluorescence signal and singlet oxygen generation when irradiated. Importantly, PpIX–GC–NPs-treated mice presented prolonged blood circulation, enhanced tumor targeting ability, and improved in vivo therapeutic efficiency in tumor-bearing mice, compared to that of free PpIX-treated mice. These results proved that this tumor-homing cellular ‘on/off’ nanoparticle system of PpIX–GC–NPs has a great potential for synchronous photodynamic imaging and therapy in cancer treatment.

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

A novel and sensitive fluorescence biosensor based on aptamer and rolling circle amplification for the determination of cocaine was developed in the present work. Here cocaine aptamers immobilized onto Au nanoparticles modified magnetic beads hybridized with short DNA strand. In the presence of cocaine, the short DNA strand was displaced from aptamer owing to cocaine specially binding with aptamer. Next, the short DNA strand was separated by magnetic beads and used to originate rolling circle amplification as primer. The end products of rolling circle amplification were detected by fluorescence signal generation upon molecular beacons hybridizing with the end products of rolling circle amplification. With rolling circle amplification and the separation by magnetic beads reducing the background signal, the new strategy was suitable for the detection of as low as 0.48 nM cocaine. Compared with reported cocaine sensors, our method exhibited excellent sensitivity. Our new strategy may provide a platform for numerous proteins and low molecular weight analytes to highly sensitively detect by DNA amplification.

An Approach to Multiplexing an Immunosorbent Assay with Antibody−Oligonucleotide Conjugates

Early detection of cancer biomarkers provides clinically valuable information. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for individual cancer markers, methods for simultaneous determination of multiple markers within a single sample are still in demand. Here, we present a novel oligonucleotide-linked immunosorbent assay (OLISA) with a multiplexing capability on the same microwell plate-based system as in ELISA. Employing a DNA oligonucleotide that is covalently conjugated to the detection antibody and a complementary RNA oligonucleotide which is appended with a fluorophore and a quencher, degradation of the RNA in the DNA-RNA duplex by RNase H is exploited for fluorescent signal generation. Iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H further lead to amplification of the detection signal in OLISA. Moreover, the use of antibody-oligonucleotide conjugates enables multiplexing of OLISA, which is successfully demonstrated by tethering DNA molecules to detection antibodies and by performing assays for three common cancer markers including α-fetoprotein, prostate-specific antigen, and carcinoembryonic antigen. With the simple procedure and reliable detection performance, the developed multiplex OLISA has a wide potential for use in analysis of a panel of biomarkers in clinical diagnostics.

Analysis of quantum dot fluorescence stability in primary blood mononuclear cells

A quantitative assessment of fluorescence signal generation and persistence in blood cells, measured at multiple points over a time course, is presented. Quantum dots (QDs) are inorganic fluorophores that are photostable and nonmetabolized and so can provide quantitative measures of cell biology over multiple cell generations. However, if the potential of these nanoparticles for long-term reporting is to be realized, an understanding of the stability of their fluorescence in living cells is essential. CdTe/ZnS and CdSe/ZnS core/shell dots with peak emission wavelengths of 705 nm and 585 nm, respectively, were loaded, via endocytosis into mononuclear cells extracted from primary blood and flow cytometry used to measure the average fluorescence intensity per cell within populations >10⁴. Time-based study showed a saturation-limited uptake of QDs with a characteristic time of 20 min and a maximum fluorescence signal that is linearly proportional to dot solution concentration. The fluorescence signal decreases after attachment and internalization within cells and is accurately described by a biexponential decay with a rapid initial decay followed by a much slower signal loss with characteristic times of 435 and 7,000 min respectively. Comparison with control samples indicates that interaction with the culture media is a major contributory factor to the initial signal decay. These results provide phenomenological descriptions of the evolving QD fluorescence within live cells with associated analytical equations that allow quantitative assessment of QD-based assays.

In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope

Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW fs laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 μm diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 h after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.

Fluorescence Aptameric Sensor for Strand Displacement Amplification Detection of Cocaine

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement. Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double-stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical.

Transillumination fluorescence imaging in mice using biocompatible upconverting nanoparticles

We report on a systematic study of upconverting fluorescence signal generation within turbid phantoms and real tissues. An accurate three-point Green's function, describing the forward model of photon propagation, is established and experimentally validated. We further demonstrate, for the first time to our knowledge, autofluorescence-free transillumination imaging of mice that have received biocompatible upconverting nanoparticles. The method holds great promise for artifact-free whole-body visualization of optical molecular probes.

An experimental and theoretical study of DAF‐FM activation by NO: Toward calibration of an NO‐sensitive fluorescent dye

The quantitative measurement of NO in vitro has been hampered by the lack of a method that can detect nanomolar levels with spatial and temporal resolution and without interference from other reaction byproducts. 4,5 diaminofluorescein (DAF‐FM) is a sensitive NO fluorescence probe used widely for qualitative but not quantitative assessment of cellular NO production. With the aid of a mathematical model of NO/DAF‐FM reaction kinetics, we have conducted experimental studies to calibrate the fluorescence signal and estimate NO generation in different systems. Kinetic analysis suggests the following dependence of DAF‐FM triazole (DAF‐T) generation rate:DAF‐T fluorescence was measured in a series of experiments with varying DAF‐FM and NO donor concentrations (spermine/NO). Experiments showed that the slope of fluorescent intensity is proportional to [NO]2 and exhibits a Monod type dependence on DAF‐FM concentration, thus validating the proposed kinetic mechanism. A calibration procedure was formed and applied in a cell culture system. Human umbilical vein endothelial cells (HUVEC) were stimulated with hemodynamic and agonist stimuli and the release rate of NO was assessed.[Supported by AHA grant NSDG043506N and NIH grant HL095101]

PHAPI/pp32 Suppresses Tumorigenesis by Stimulating Apoptosis

PHAPI/pp32 is a tumor suppressor whose expression is altered in various human cancers. Although PHAPI possesses multiple biochemical activities, the molecular basis for its tumor-suppressive function has remained obscure. Recently we identified PHAPI as an apoptotic enhancer that stimulates apoptosome-mediated caspase activation. In this study, we defined the structural requirement for its activity to stimulate caspase activation using a series of truncation mutants of PHAPI. Further, utilizing these mutants, we provide evidence to support the model that the apoptotic activity of PHAPI is required for its tumor-suppressive capability. Consistently, pp32R1, a close homolog of PHAPI and yet an oncoprotein, is not able to stimulate caspase activation. A highly discrete region between these two proteins localizes to an essential caspase activation motif of PHAPI. Additionally, PHAPI is predominantly a nuclear protein, and it can translocate to the cytoplasm during apoptosis. Disruption of the nuclear localization signal of PHAPI caused a modest decrease of its tumor-suppressive function, indicating that nuclear localization of PHAPI contributes to, but is not essential for, tumor suppression.

Two-color two-photon excitation of indole using a femtosecond laser regenerative amplifier

The fluorescence emission from indole resulting from two-color two-photon (2C2P) excitation with 400 and 800nm wavelengths is observed, using the second harmonic and fundamental wavelength of a 800nm 40fs pulsed Ti:Sapphire femtosecond (fs) regenerative amplifier operating at a repetition rate of 1kHz. By delaying one fs laser pulse relative to the other, the cross correlation of fluorescence is observed, which indicates the generation of 2C2P fluorescence signal in the experiment. The strongest 2C2P fluorescence emission characterized by the peak of cross correlation curve suggests optimal temporal overlap of the two fs laser pulses. The 2C2P fluorescence signal is linearly dependent on the total excitation intensity. The fluorescence signals with 400nm and 800nm irradiation alone are also demonstrated and discussed in this paper.

Amplification of microsphere-based microarrays using catalyzed reporter deposition

Assay sensitivities using three fluorescent signal generation schemes were evaluated on the Luminex flow cytometer. Following microsphere capture of antigen by immobilized antibodies, bound targets were quantified by use of (1) Cy3-labeled “tracer” antibodies (30min total time), (2) biotinylated tracers followed by streptavidin-R-phycoerythrin (60min total time), or (3) biotinylated tracers followed by avidin-peroxidase conjugates and tyramide signal amplification (TSA; 90min total time). Use of TSA for signal generation in three individual toxin assays improved performance up to 100-fold over Cy3-antibody-based detection, and while streptavidin-R-phycoerythrin provided equivalent sensitivities, TSA produced dramatic increases at low concentrations simplifying positive sample identification. Detection limits for TSA-interrogated assays for ricin, cholera toxin, and staphylococcal enterotoxin B were 64pg/ml, 4pg/ml, and 0.1ng/ml, respectively, using optimized conjugates; analogous detection limits for Cy3-antibody-interrogated assays were 8ng/ml, 1ng/ml, and 1ng/ml, respectively. No improvement was observed in botulinum toxoid A assays when TSA amplification was used. As unique preferences for specific avidin-peroxidase conjugates were observed in the individual assays, improvements in multiplexed assays utilizing a single conjugate were significantly lower (3–10-fold improvements). Furthermore, increases in variability resulted in poorer performance of TSA-interrogated assays for botulinum toxoid, indicating that assay-specific optimization should be performed, especially prior to multiplexing.

Quality assurance for polychromatic flow cytometry

This protocol outlines a three-part quality assurance program to optimize, calibrate and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (a practice known as polychromatic flow cytometry). The initial steps of this program (system optimization) ensure that the instrument's lasers, mirrors and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of photomultiplier tube (PMT) voltages by determining the PMT voltage range and linearity (Steps 2-10) and validating the PMT voltage (Steps 11-17). Finally, to ensure consistent performance, we provide procedures to monitor the precision, accuracy and sensitivity of fluorescence measurements over time. All three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer's optical path. However, only a few of these procedures need to be carried out on a routine basis.

Characterizing the Secondary Structure and Identifying Functionally Essential Nucleotides of pH6DZ1, a Fluorescence-Signaling and RNA-Cleaving Deoxyribozyme

pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.