Fluorescent Labels and Techniques

Organic fluorophores contain aromatic groups and conjugated bonds, which allow for easy excitation and specific emission wavelengths.

Fluorescent proteins, such as GFP from jellyfish, are used as fusion proteins and exhibit natural bioluminescence.

Enzymes in fluorometric ELISA convert non-fluorescent substrates into fluorescent products, enabling detection.

<p>Drawbacks include a background signal, self-quenching at high concentrations, and a short fluorescence lifetime.</p> Signup and view all the answers

<p>Common lanthanide elements include Europium (Eu), Terbium (Tb), Samarium (Sm), and Dysprosium (Dy), notable for their intense fluorescence.</p> Signup and view all the answers

<p>The fluorescence signal from lanthanides is observed when they are firmly bound to an organic ligand that shields them from water.</p> Signup and view all the answers

<p>Lanthanide ions provide intense fluorescence, a wide stoke shift, sharp emission bands, and long-lived fluorescence.</p> Signup and view all the answers

<p>Non-fluorescent chelates are structures that bind lanthanide ions but do not emit fluorescence themselves.</p> Signup and view all the answers

<p>The AEB analogue increases as the number of 'on beads' rises.</p> Signup and view all the answers

<p>AEB digital is defined as the negative natural logarithm of 1 minus 'on beads'.</p> Signup and view all the answers

<p>Digital ELISA provides better sensitivity than chemiluminescence or analogue fluorescence.</p> Signup and view all the answers

<p>Different analytes are distinguished using color-coded, fluorescence-labeled beads.</p> Signup and view all the answers

<p>Tracer binding to solid phase and cross-reacting substances blocking the antibodies.</p> Signup and view all the answers

<p>A reaction requires that something must be reduced and something must be oxidized.</p> Signup and view all the answers

<p>Cyclic voltammetry or amperometry are used to measure current changes.</p> Signup and view all the answers

<p>In electrochemical ELISA, the antibody is conjugated with HRP enzyme and requires oxidizable substrates.</p> Signup and view all the answers

<p>Inherently fluorescent chelates do not require a development step, thus simplifying the assay process.</p> Signup and view all the answers

<p>The acidic buffer's purpose is to dissociate the ions, facilitating the formation of new fluorescent chelates.</p> Signup and view all the answers

<p>High fluorescence and good stability are two key criteria for an effective lanthanide chelate reporter.</p> Signup and view all the answers

<p>The presence of EDTA in blood plasma can interfere with the assay, making it unsuitable for this sample type.</p> Signup and view all the answers

<p>Time-resolved fluorescence allows for long-lived fluorescence, enabling precise timing in measurement for enhanced sensitivity.</p> Signup and view all the answers

<p>Direct TRF assays do not require a washing step, unlike DELFIA which requires careful washing before measurement.</p> Signup and view all the answers

<p>Triton X-100 is included to stabilize the chelates and form hydrophobic pockets to shield them from water.</p> Signup and view all the answers

<p>Measurement timing must be precise; otherwise, it can lead to inaccurate signal readings.</p> Signup and view all the answers

<p>The conjugate application buffer facilitates the addition of the conjugate by providing necessary agents like proteins, polymers, and surfactants.</p> Signup and view all the answers

<p>The wicking pad pulls fluid upstream through the strip and prevents back flow, acting as the engine of the assay.</p> Signup and view all the answers

<p>Backing materials are typically made of plastic, commonly polystyrene, and are coated with pressure sensitive adhesive.</p> Signup and view all the answers

<p>An optimal analytical membrane must allow liquid movement through capillary forces and bind proteins irreversibly on test and control lines only.</p> Signup and view all the answers

<p>Nitrocellulose membranes face high non-specific binding due to their high protein binding capacity and variability in performance under different environmental conditions.</p> Signup and view all the answers

<p>Larger pore sizes allow for faster liquid flow but may compromise the sensitivity of the assay due to reduced interaction opportunities with target proteins.</p> Signup and view all the answers

<p>Polyvinylidene fluoride uses hydrophobic forces, while Nylon operates through ionic electrostatic forces.</p> Signup and view all the answers

<p>Flow time is defined as the number of seconds it takes for liquid to cross 4 centimeters of the membrane.</p> Signup and view all the answers

<p>The threshold cycle indicates the point at which the signal surpasses the background, with a higher quantity of analyte leading to a quicker approach to this cycle.</p> Signup and view all the answers

<p>It may bind to non-target sequences like primer dimers, resulting in inaccurate measurements.</p> Signup and view all the answers

<p>The ratio of adenine-thymine (A-T) to guanine-cytosine (G-C) pairs, ensuring no secondary structures, and a length between 18 to 24 nucleotides.</p> Signup and view all the answers

<p>The probe binds to the target sequence, separating the reporter from the quencher, which allows the signal to be detected.</p> Signup and view all the answers

<p>They exist in a hairpin structure that opens during denaturation, allowing the reporter to bind to the amplicon while being separated from the quencher.</p> Signup and view all the answers

<p>They enhance signal strength through FRET by having a donor and an acceptor probe that are positioned closely during annealing.</p> Signup and view all the answers

<p>These repeats can lead to mispriming or the formation of secondary structures, compromising the efficiency of the PCR.</p> Signup and view all the answers

<p>TaqMan probes undergo hydrolysis during amplification, resulting in the separation of the fluorophore from the quencher, which generates a measurable signal.</p> Signup and view all the answers