Fluorescent Dyes

Application of Fluorescent Probes

Introduction to fluorescent probes:

After being excited by the excitation light, it returns to the ground state from the excited state singlet state, and has characteristic light emission in the ultraviolet-visible-near-infrared region, which is called fluorescence. A class of fluorescent molecules whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can be sensitively changed with the properties of the environment, such as polarity, refractive index, viscosity, etc., are called fluorescent probes. There are many types of fluorescent probes, which can be divided into organic and inorganic probes according to material properties, molecular probes and nanoprobes according to the size of the probes, and single-photon, two-photon and multi-photon fluorescent probes according to the excitation light source. The needles can also be classified into metal ion fluorescent probes and biomolecular fluorescent probes according to the analyte. Fluorescent probes are widely used in various detection and labeling, such as determination of metal ions, pesticide residues, biomolecule content, tracer biomolecules, labeling of macromolecules and cellular and subcellular structures.

Fluorescent probes are described in many cases as fluorescent chemosensors. Fluorescent probes are small molecules that have the ability to absorb light at specific wavelengths and emit light at different wavelengths, usually longer wavelengths (a process called fluorescence), used for Study biological samples. These molecules, also known as fluorophores, can be attached to target molecules and used as labels for analysis by fluorescence microscopy.

The fluorescent molecules of a fluorophore respond significantly to light. Each fluorophore has different characteristics that can be used to determine which fluorophore to use for a given application or experimental system. Some proteins or small molecules in cells are naturally fluorescent; this is called intrinsic fluorescence or autofluorescence [eg, green fluorescent protein (GFP)]. Proteins, nucleic acids, lipids, or small molecules can be labeled with an extrinsic fluorophore (a fluorescent dye), which can be a small molecule, protein, or quantum dot. This article discusses the pitfalls and problems of various fluorescent compounds currently in use.

Fluorescent probes have received increasing attention in molecular biology research and development. Many scientists are studying these dazzling natural wonders in fields such as medicine, pharmaceuticals and green biotechnology.

Fluorescent Probe Definition

The term “probe” is often used to describe fluorescent oligonucleotides. The most commonly used DNA probes are double-labeled and therefore consist of three distinct parts:
※ Single-stranded DNA between 5 and 35 bp responsible for specific DNA recognition
※ Reporter dyes that emit fluorescence at specific wavelengths
※ A quencher that absorbs the light emitted by the reporter dye in a certain state.

DNA probes are labeled with an appropriate reporter dye and quencher pair to take advantage of Frster resonance energy transfer (FRET for short).

Let’s take a closer look at the other side of fluorescence:

Fluorescence is defined as “the spontaneous emission of light in response to light illumination”. In other words, the electrons enter a higher energy state upon photoexcitation. However, this state is highly unstable.
Once the excited electron returns to a lower energy state, the excess energy is emitted in the form of photons.
Fluorophores come in all the colors of the rainbow, depending on the energy they emit.

For short DNA probes, separation of the reporter dye and quencher occurs after the polymerase hydrolyzes the probe and releases the reporter dye.

Probes that require hydrolysis are called TaqMan probes and are based on TaqMan polymerase with 3′-5′ exonuclease activity.

Use of fluorescent probes:

One of the main uses of probes is to visualize target DNA sequences for quantitative real-time PCR (qPCR), which is an extremely versatile tool thanks to fluorescent probes.
Quantitative PCR can also be performed using fluorescent dyes that bind to dsDNA, the amount of which increases during PCR. However, these dyes bind to any dsDNA and do not distinguish the target DNA from other DNA strands. Conversely, probes show higher specificity because specific oligonucleotide moieties anneal only to the target sequence.
Hybridization probes allow for further applications, in addition to pathogen quantification, mutation detection, melting point analysis, and in situ hybridization.
Additionally, probes can be labeled with different reporter dyes, allowing multiplex PCR to detect multiple targets in a single reaction.