Bio-optical marker refers to marking biomolecules with markers with optical characteristics, so as to achieve the purpose of detection and identification. According to the different optical characteristics of the application, it can usually be divided into fluorescent molecular labeling, fluorescent protein labeling, bioluminescent labeling, chemiluminescent labeling, etc. According to the purpose of marking, it includes biomolecular markers, biochemical markers, cytological markers, morphological markers and so on.
The detection of life molecules, the observation of life processes, and the identification of specific biological tissues are usually difficult to directly observe due to their microscopic and hidden nature, and even beyond the detection range of the instrument. It can ‘stand out’ and be detected with the help of the corresponding instruments.
Label the specific biomolecules, cells or tissues that current detection methods can detect and detect the molecules and nanoparticles, and then obtain the characteristics of the specific molecules, cells or tissues by detecting the number and distribution of the molecules/particles, and then obtain a certain area As well as the response and signal of molecular, biochemical and physiological indicators in cells or organisms. This labeling process is commonly referred to as Biomarker. Bio-optical marker refers to marking biomolecules with markers with optical characteristics, so as to achieve the purpose of detection and identification. According to the different optical characteristics of the application, it can usually be divided into fluorescent molecular labeling, fluorescent protein labeling, bioluminescent labeling, chemiluminescent labeling, etc. According to the purpose of marking, it includes biomolecular markers, biochemical markers, cytological markers, morphological markers and so on.
Bio-optical markers, because of the use of optical methods, can achieve high contrast, high resolution, high sensitivity, high signal-to-noise ratio, and convenient and quick detection with the help of mature optical high-sensitivity detection instruments. chosen as the primary biomarker modality. Especially after the discovery of fluorescent proteins, bio-optical labeling has achieved rapid application development. Osamu Shimomura, Martin Charfie and Roger Tsien won the 2008 Nobel Prize in Chemistry for their discovery and research on green fluorescent protein. Bio-optical labels are now widely used as tracers in life science and medical research.
History of Bio-optical Labeling Research and Application
In biological research, scientists often use fluorescent molecules that emit fluorescence as biomarkers. By chemically attaching this fluorescent molecule to other invisible molecules, the previously invisible part becomes visible. Biologists have been using this labeling method to “pull” otherwise transparent cells or organelles out of the dark microscope field of view.
When traditional fluorescent molecules emit light, they will generate toxic oxygen free radicals, causing the observed cells to die, which is called “phototoxicity”. Therefore, before the discovery of green fluorescent protein, scientists could only study by fluorescent labeling. The static structure of dead cells, or their toxic effects have to be temporarily ignored, and live cells are only observed for a short time, while the phototoxicity of fluorescent proteins is very weak, which is very suitable for labeling various living cells.
The fluorescent protein was first discovered in 1962 in a jellyfish named Aequorea victoria. The protein produced by its gene emits green fluorescence when excited by blue wavelength light. The light-emitting process also requires the help of the light-emitting protein Aequorin, and this protein also needs to interact with calcium ions (Ca).
The phototoxicity of GFP is very weak, making it ideal for labeling living cells. However, it took more than 20 years after the discovery of green fluorescent protein that it was used to label biological samples. In 1993, Martin Schalfi successfully made other organisms (such as Escherichia coli, etc.) other than jellyfish to produce green fluorescent protein through genetic recombination. He not only confirmed the compatibility of green fluorescent protein with living organisms, but also established the basic method of using green fluorescent protein to study gene expression, and many modern major diseases are related to abnormal gene expression.
Later, Chinese-American Qian Yongjian systematically studied the working principle of green fluorescent protein, and carried out drastic chemical transformation on it, which not only greatly enhanced its luminous efficiency, but also developed red, blue, and yellow fluorescent proteins. , making fluorescent proteins truly a toolbox for biologists to choose as needed. Most of the fluorescent proteins commonly used in biological laboratories are variants modified by Qian Yongjian.
With these fluorescent proteins, using optical instruments, scientists seem to have installed “lighthouses” in cells, allowing them to monitor various life processes in real time. Through Sharfi’s idea of gene cloning, scientists have so far cultivated fluorescent mice and fluorescent pigs.
In addition, in addition to the above-mentioned fluorescent molecules and fluorescent protein labels, since 2000, with the development of bio-nanotechnology, some new and biocompatible optical nano-labels have also been researched and developed, such as upconversion nanoparticles, quantum Dots, long-time-lapse fluorescent particles, etc. have all been studied and utilized. It is used as a biological marker of cells and living bodies, as a powerful weapon for “sensing” of biological optical imaging.
Types and applications of biological optical markers
Fluorescent molecular/nanoparticle marker
Fluorescent molecules include organic reagents or metal chelates; fluorescent nanoparticles include upconversion, quantum dots, etc. It has strong characteristic fluorescence in the ultraviolet-visible-near-infrared region. After labeling cells and living bodies with this molecule/nanoparticle, optical tracer detection can be realized, or the excitation and emission wavelength, intensity, Fluorescence properties such as lifetime and polarization can be sensitively changed with changes in environmental properties such as polarity, refractive index, and viscosity, and biological detection can be performed using this property. Fluorescent molecule/nanoparticle labeling is flexible in design and convenient in application.
Bioluminescence labeling is a biological labeling method that uses luciferase (Luciferase) gene to label cells or DNA. After labeling, the cells synthesize the luciferase, and then add exogenous luciferase. Below, luciferin emits light after oxidation. The method allows researchers to directly monitor cellular activity and gene behavior in living organisms. Through this system, biological processes such as tumor growth and metastasis, the development of infectious diseases, and the expression of specific genes in living animals can be observed. Because of its extremely simple operation, intuitive results and high sensitivity, it has been widely used in life sciences, medical research and drug development.
Fluorescent protein marker
After the fluorescent protein gene fragment is connected with the target gene, it is transfected into the cell, and after it is expressed normally, it can be observed and detected by fluorescence microscope, flow cytometer or laser confocal microscope under excitation light. Fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and yellow fluorescent protein (YFP). It can be harmless to live cells and can be observed for a long time. Therefore, it is widely used in the research of transgenic animals, fusion labeling, gene therapy, protein functional localization and migration changes in living cells, molecular process of pathogenic bacteria invading living cells, etc. Fluorescent proteins are attracting more and more attention in life science research as a new generation of gene transfer reporters and/or localization markers
A method of attaching a compound that activates luminescence to a molecule (protein, nucleic acid, etc.) to be detected. It is also possible to connect haptens (such as digoxigenin, biotin, etc.), and then combine with enzyme-labeled anti-hapten antibody or avidin, and bind to the enzyme-labeled antibody or avidin on the hapten. Can catalyze chemical changes in chemiluminescent substrates to emit light. For example, the antibody molecule is labeled with acridinium ester, and it emits light after being activated by a trigger, which is used to detect the solid-phase antigen.
With the help of rapidly developing optical detection technology and mature optical imaging instruments, bio-optical markers have shown strong application vigor from the very beginning. It has a wide range of applications in various fields such as molecular detection, biological mechanism research, and tumor marker diagnosis. Bio-optical labeling technology opens a window for people to observe the mysterious microscopic biological world, allowing people to truly “see” biological processes and life phenomena that were previously unseen. Bio-optical technology is in the ascendant, and the application of bio-optics needs to be further developed. It is believed that with the development of bio-optical technology, people will be able to track and observe life processes on a smaller scale and more accurately.