Fluorescent probes have characteristic fluorescence in the ultraviolet-visible-near infrared region, and their fluorescent properties (excitation and emission wavelength, intensity, lifetime, polarization, etc.) can be changed according to the nature of the environment, such as polarity, refractive index, viscosity, etc. A class of fluorescent molecules that change sensitively. Non-covalent interactions with nucleic acids (DNA or RNA), proteins, or other macromolecular structures that change one or more fluorescent properties. Can be used to study the nature and behavior of macromolecular substances.
Figure 1. Fluorescence of different substances under UV light.
There are two types of fluorescent chemicals used in fluorescent quantitative PCR: fluorescent probes and fluorescent dyes. During PCR amplification, a specific fluorescent probe is added at the same time as adding a pair of primers. The probe is an oligonucleotide, and the two ends are respectively labeled with a reporter fluorescent group and a quenching fluorescent group. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3 'exonuclease activity of the Taq enzyme digests and degrades the probe, making the reporter fluorophore and The fluorescing group is separated, so that the fluorescence monitoring system can receive a fluorescent signal, that is, each amplified DNA strand, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
It is most commonly used for labeling antigens or antibodies in fluorescent immunoassays. It can also be used for micro-environment detection, such as surfactant micelles, bilayer membranes, and protein microsites. Probes are usually required to have large molar absorption coefficients and high fluorescence quantum yields; fluorescence emission wavelengths are in long wavelengths with large Stokes shifts; when used in immunoassay, binding to antigens or antibodies should not affect their activity. It can also be used to label pending nucleotide fragments and specifically and quantitatively detect the amount of nucleic acid.
Figure 2. A fluorophore-labeled human cell.
Commonly used fluorescent probes include fluorescein-based probes, inorganic ion fluorescent probes, fluorescent quantum dots, molecular beacons, and the like. In addition to the quantitative analysis of nucleic acids and proteins, fluorescent probes are widely used in nucleic acid staining, DNA electrophoresis, nucleic acid molecular hybridization, quantitative PCR technology, and DNA sequencing.
Methods for detecting fluorescent probes include single-point measurement and charge-coupled device (CCD) fluorescence imaging (including laser confocal fluorescence microscopy for micro-area analysis). Due to the long scanning time of the photomultiplier tube and the high intensity of laser irradiation, it is difficult to catch the early changes in fluorescence. The CCD fluorescence imaging has a large area array, a wide imaging field, and adjustable imaging time, so the detection effect is better.
CD BioScience provides a series of probes that can be used to detect different substances, including oxygen probes, potentiometric probes, chemotaxis probes and transferrin probes.