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Synthesis and Chemiluminescence of Luminol - YouTube

125. Bertoncello P. Nanomaterials for biosensing with electrochemiluminescence (ECL) detection. 2011;16:1084-108

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Luminol Synthesis and Chemiluminescence Flashcards | …

Fluorescent burst coincidence detection is designed specifically for SMD platforms. Two QD labeled probes were designed to recognize the same target and form a dually labeled complex (Fig. A). As the complex passes through the detection volume, signals from both QDs are detected simultaneously, resulting in a pair of coincident fluorescent bursts in two independent channels (Fig. B). The concentrations of probes and targets are in the sub-nanomolar range. Under such conditions, the average number of molecules remaining in the detection volume is less than unity. As a consequence, in the absence of target, two probes pass through the detection volume independently and the resulting fluorescent bursts in each of the two channels are uncorrelated (Fig. C). It is important to ensure that no spectral crosstalk exists between the two fluorophores to avoid false coincidence. Due to small Stokes shift of organic dyes, dual-excitation is required for coincidence detection. However, it is intricate to align the illumination volumes of two lasers and correct the chromatic aberration []. In contrast, the unique optical properties of QDs permit single excitation for QDs of different colors. As a result, multi-color coincidence can be achieved with QDs using simple confocal setups with a single excitation source.

Synthesis and Chemiluminescence of Luminol - …

Despite their numerous advantages, it is to be realized that QD is not meant to replace conventional organic fluorophores but rather offer a complement. Both dyes have their benefits as well as drawbacks. The sizes of QDs are considerably larger than typical organic fluorophores, which poses a problem for QD conjugated probes in biochemical reactions. QDs also unsuitable for many enzyme based signal amplification reactions []. So far, no practical QD based real time PCR platform has been reported that match conventional organic fluorophore based real time PCR in terms of performance. Concerns are also raised on the cytotoxicity of QD for studies. The majority of QDs are made from highly toxic semiconductor materials. Even with proper capping and organic functional layers, toxic ions are still believed to escape from the core []. Fortunately, the abovementioned issues are already under investigation. Compared to organic fluorophores, more detailed characterization and well established assay protocols are required to promote the commercial availability of QD based analysis systems in order to attract more users.

Synthesis And Chemiluminescence Of Luminol Lab ..

Since their introduction in biological applications merely a decade ago, QDs have quickly evolved from a generic "passive" fluorescent labels to "smart" nanoprobes that carry additional functions. QDs have made great impact on modern molecular and cellular biology by providing innovative tools to explore new biological events. Their unique optical properties render them valuable for high throughput and multiplexed detection, particularly in -omics studies. QD-FRET based nanosensors have been integrated into a great number of homogeneous molecular assays to detect specific targets and monitor reaction progress. QDs have also proven to be excellent tags for SMD strategies due to their extreme sensitivity. Unconventional non-photoluminescence properties of QDs are already being explored in the hopes of developing new detection methods.

QEMSA working principle. A) Biotin-tagged DNA fragments were generated from genomic DNA targets using biotinylated primers and a limited number of amplification cycles to preserve genomic DNA quantity information. The biotinylated DNA fragments were then mixed with streptavidin-coated QDs, and self-assembly would occur to form nanocomplexes where the resultant DNA:QD ratio, N, was dependent on the amount of input DNA. The electrophoretic mobility of the nanocomplexes increased with the DNA:QD ratio and was used to determine DNA quantity. B) Pseudocolor gel image reveals that the QDDNA nanocomplexes (combined green and red) migrated faster than the naked QDs (green) but slower than the oligonucleotides alone (red). C) Representative gel image of QDDNA nanocomplexes with various N values migrating in an agarose gel. The nanocomplexes with the largest N migrate fastest and vice versa. D) Migration curve was obtained by plotting the migration distance of each gel band against the respective DNA:QD ratio, N. The migration distance was determined by measuring the point at which the leading edge of the electropherogram met the baseline intercept. Reprinted with permission from [], copyright 2011 American Chemical Society.

Luminol Synthesis of a Chemiluminescent Substance - …

85. Freeman R, Liu X, Winner I. Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer-substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots. 2011;133:11597-604

With the development of novel fluorescent probes and sensing strategies, SMD pushes the detection limit to the extreme, providing an exceptional platform for sensing scarce molecules at low concentrations. QDs are ideal fluorescent tags for SMD based sensing. The signal to noise ratio of SMD is highly dependent on specific brightness of the fluorophores and the transient time of molecules passing through the detection volume. To attain high analysis throughput, samples are often driven through the detection volume at high speed, which decreases the molecule transient time and inevitably reduces the burst hence the signal to noise ratio. QDs alleviate these issues due to their high brightness. In addition, QDs are exceedingly photostable when compared to organic fluorophores, which allow them to withstand high-intensity illumination within the confocal setup for much longer periods of time.

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In addition to environmental conditions, QD emission intensities are strongly influenced by proximal molecules or nanoparticles that QDs interact with. Under many circumstances, the photoluminescence of QDs is drastically quenched through numerous mechanisms. This seemingly undesirable phenomenon can serve as an advantageous feature. For example, quenching mechanism can be designed to act like a molecular switch for fluorescent signals, which would make QDs an ideal homogeneous sensing platform for studying molecular interactions and detecting specific targets.

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Single molecule detection (SMD) techniques have made great advancements in past few decades. Solution phase SMD is developed primarily based on confocal fluorescence spectroscopy or microscopy, which acquires signal from a confined volume (typically femtoliter range). The detection volume is limited by the illumination volume of the laser source and the collection efficiency function confined by the pinhole []. SMD offers tremendous advantages over conventional fluorescence based detection methods. The small detection volume greatly reduces the background noise thereby improving detection sensitivity. While performing SMD measurements, the targets are kept at low concentrations so that the average number of molecules residing in the detection volume is less than unity. The passage of target molecules through the detection volume results in single fluorescent bursts carrying the information of individual molecules. Unlike ensemble analysis that measures averaged fluorescence properties, SMD interrogates individual molecules and provides statistical information on the entire target population.

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Chemiluminescence is another alternative energy source that serves as a FRET donor. For example, Luminol is a chemiluminescent reagent that is activated by oxidants such as H2O2. The reaction requires a catalyst to decompose H2O2 into H2O and O2. The hemin/G-quadruplex horseradish peroxidase (HRP) mimicking catalytic nucleic acids (DNAzymes) were discovered to generate chemiluminescence through catalyzing the oxidation of luminol by H2O2 [, ]. Freeman included aptamer domains into the DNAzyme subunits []. One of the aptamer subunits was conjugated to a QD. In the presence of aptamer targets, ATP or Hg2+ in this case, the DNAzyme subunits self assembled into active hemin/G-quadruplex DNAzyme structures and promoted the chemiluminescence resonance energy transfer (CRET) by catalyzing luminol emission. As shown in Figure A, nucleic subunits included domain I and II of the HRP mimicking DNAzyme, as well as domain V and VI of an anti-ATP aptamer. In the absence of ATP, the two subunits are not able to form a stable complex. However, in the presence of ATP, the aptamer domains binds to ATP and the resulting complex leads to the formation of a hemin/G-quadruplex that catalyzes the chemiluminescent reaction and gives rise to CRET. In contrast to FRET, the emission intensities of donors and acceptors increased or decreased concurrently because the amount of energy transferred to the QD was proportional to the chemiluminescent energy available (Fig. B). QD-CRET sensors were also configured to detect specific DNA sequences (Fig. C). A DNA hairpin structure consisting of a few functional domains were conjugated to QDs. The DNAzyme forming domain was blocked in the presence of the hairpin loop. The sequence recognition domain resided in the loop. As the target DNA hybridized to the recognition sequence and opened the hairpin, the DNAzyme forming domain was freed, leading to the self assembly of a hemi/G-quadruplex DNAzyme. DNA hairpins with three different target recognition sequences were conjugated to QD490, QD560 and QD620 respectively to form three QD-CRET DNA probes. Upon hybridization to their respective targets, Hemin and H2O2 were added to induce CRET. The presence of targets was indicated by emission of specific QDs probes through CRET. With the proposed QD-CRET sensor, the authors successfully resolved three targets in a multiplexed format (Fig. D).

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