Bioconjugation in Biomolecular Labeling and Imaging

The field of biomolecular labeling and imaging is closely related to the use of bioconjugation for assay and quantification, but in this case, the aim is to reveal the presence or location of the target molecule without necessarily quantifying it. Approaches in this field typically involve the use of bioconjugates consisting of targeting components coupled to detection components to identify biomolecules in cells, tissues, or organisms. It also includes semi-quantitative analysis of specific biomolecules after electrophoretic separation and blotting, such as chemiluminescence detection of Western blots using antibody-enzyme conjugates. The use of bioconjugates for detection, tracing, and imaging has driven the rapid development of techniques such as immunochemical staining, fluorescence microscopy, and high-content analysis (HCA) that are critical to improving the understanding of cell biology. They also constitute the most important new technology platform for in vivo imaging for diagnostic or therapeutic purposes.

Used to detect, track, or image the most commonly used target molecules in cells or tissue is required for target molecules with specific antibodies or antibody fragments. Now tens of thousands have clear specific antibodies commercially available, and many of these antibodies have proven data, proving the practicability of them for a specific application. Combining such antibodies with appropriate detection molecules could facilitate their use in specific imaging techniques. Often, however, primary antibodies are too small in quantity and too expensive to economically prepare conjugates with them. In this case, two resistance coupling can be used to detect resistance in imaging applications. A convenient alternative to making primary antibody conjugates is to use biotinylated primary antibodies, which can then be detected using labeled streptavidin conjugates. The use of secondary antibody conjugates or streptavidin conjugates is the most popular strategy for the detection of primary antibodies in detection, tracing, and imaging applications.

Advantages and Features

Bioconjugation offers numerous advantages and distinctive features that make it indispensable for biomolecular labeling and imaging. One of the foremost benefits is its ability to provide high specificity and sensitivity in detecting target molecules. By using specific probes that bind selectively to their targets, researchers can achieve precise labeling with minimal background noise. This specificity is crucial in applications such as immunohistochemistry, where accurate localization of antigens is essential for meaningful interpretation.

Another key advantage is the versatility of bioconjugation. It can be applied to a wide range of biomolecules, including proteins, peptides, nucleic acids, and small molecules. This adaptability allows for the creation of diverse conjugates tailored to various experimental needs. Moreover, bioconjugation reactions can be optimized to preserve the biological activity of the labeled molecules, ensuring that the functional properties of the biomolecules are maintained post-labeling.

Bioconjugation also facilitates multiplexing, where multiple targets can be labeled and detected simultaneously within a single sample. This capability is particularly valuable in complex biological studies, enabling the simultaneous analysis of multiple biomarkers and their interactions. Additionally, conjugated probes can be designed to emit signals in different spectral regions, allowing for the use of advanced imaging techniques such as fluorescence resonance energy transfer (FRET) and multiphoton microscopy.

Conjugation Strategies

The working principle of bioconjugation involves a series of well-defined chemical reactions that covalently link imaging molecules to biomolecules. The choice of bioconjugation chemistry depends on the nature of the biomolecule and the desired application.

Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate（SMCC）

Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) is a kind of containing N-hydroxy succinimide ester and maleic imide (NHS) activity of bifunctional coupling agent, which can be respectively containing sulfydryl and amino compounds keyed in together.

SMCC activated protein (enzyme/fluorescein) process is called Malay acylation, is the first protein amino connects to SMCC, forming half coupling. Then blended with antibodies to coupling, completes the antibody and protein coupling, the coupling structure needs to be antibody of disulfide bond must back into free mercaptan (sh) to react with maleic imide.

SMCC is difficult to dissolve in water, so SMCC must be dissolved in organic solvents such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) before protein modification. In some cases, even a small amount of organic solvents will affect the activity of the protein. Generally, organic solvents should not exceed 10% of the aqueous medium, otherwise, the activity of the protein or drug will be affected. The sulfonated derivative, sulfo-SMCC, has a negatively charged sulfonic acid group on the succinimide ring, which increases its water solubility.

The advantage of this method is that it does not destroy the antigen-antibody binding site. However, the disadvantage is that this method is usually autoreactive since proteins often contain amine and thiol groups, which cause significant self-cross-linking.

Example of SMCC (Wei W., et al., 2020)

Buccutite

Buccutite provides the most convenient and effective crosslinking method, two kinds of biological molecules can be wired together, and has a very high coupling efficiency. The method USES a pair of crosslinking agents: Buccutite MTA and Buccutite FOL. Crosslinking the MTA on the antibodies, and crosslinking FOL into protein (enzyme/fluorescein). Coupling reactions were initiated by mixing Ab-Buccutite MTA and protein-Buccutite FOL. The coupling reaction was carried out under extremely mild neutral conditions without any catalyst.

This method significantly enhanced stability compared to the SMCC coupling method. The Buccutite method is fast, the reaction is in the PH range of 5-9 for 1-2 hours to form a stable antibody fluorescent conjugate. Additionally, the Buccutite method has high coupling efficiency, it can effectively avoid self-cross-linking of antibodies or proteins, so as to have higher labeling efficiency. But the coupling position of the Buccutite method is not fixed, SMCC method in tandem antibody fragments of FC(Fragment, crystallizable).

Click Chemistry

Click chemistry-mediated coupling has been increasingly used to develop new molecular imaging probes, and radiolabeling by click chemistry-mediated reactions is bidirectional. Among various click chemistry reactions, the 1, 3-dipolar cycloaddition reaction (CuAAC) between azide and alkyne catalyzed by Cu(I) has been frequently used to develop radiomedicine. F-labeled small molecule probes prepared by CuAAC reaction are widely used and evaluated in clinical Settings. But in the development of the radioactive nuclide metal immune PET probe, had to give up using Cu (I) catalyst, a common alternative, azide - acetylene cycloaddition reaction (SPAAC), not need the catalyst can be conducted to application in radioactive nuclide metal tag way.

However, the complexity of octyl precursor in SPAAC system synthesis and hydrophobic may limit its wide application. Trans cyclooctene (TCO) and four electron-deficient oxazines (Tz) between the electronic demand Diels - Alder (IEDDA) reaction is a biological orthogonal chemical reactivity and application possibilities in areas such as a big step forward. Thus, this chemical approach has been widely used to develop molecular imaging probes.

Example of Click chemistry R1=Antibody, R2= Chelating agent (Wei W., et al., 2020)

Enzyme-Mediated radiolabeling

The enzyme-mediated method is very suitable for site-specific labeling of antibody carriers. One of the most prominent is the use of sortase A(SrtA), an enzyme mainly derived from Gram-positive Staphylococcus aureus. Typically, SrtA recognizes a substrate containing a C-terminal LPXTG motif (where X represents any amino acid except proline) and cleans the peptide between threonine (Thr) and glycine (Gly), resulting in loss of the downstream portion of the substrate (e.g., his tag) and formation of a new peptide bond with a nucleophilic substrate containing an N-terminal glycine residue. SrtA is a mature enzyme responsible for anchoring LPXTG-containing proteins to the growing cell walls and pili of various Gram-positive bacteria, while recombinant SrtA has developed in recent years as a valuable protein engineering tool. By using SrtA transformation, it is easy to functional parts (such as chelating agent and dye) installed to the antibody of N - or C - end.

Example of Enzyme-Mediated radiolabeling (Wei W., et al., 2020)

Dock-and-Lock method

To further advance the clinical translation and application of pretargeted imaging and therapeutics, a more innovative approach (Dock-and-Lock) is being used to develop humanized recombinant Bsab at scale. An example is the Anti-CEA and Anti-HSG TF2 BsAb, which contains a humanized Fab fragment from the Anti-HSG mab 679 and two humanized Anti-CEA Fab fragments from the hMN14 mab rabetuzumab.

As hapten peptides in pretargeting systems, radiolabeled small molecules possess two HSG groups and various chelating agents, conditions that allow their multifunctional labeling with different radionuclides of interest. Currently, several other such Bsab have been generated by this method.

Applications in Biomolecular Labeling

Bioconjugation is extensively utilized in the labeling of biomolecules, enabling researchers to investigate biological processes with unparalleled precision. Here are some prominent applications:

Protein Labeling: Proteins can be labeled with fluorescent dyes, enzymes, or biotin to facilitate their detection and quantification. Fluorescently labeled proteins are widely used in techniques such as western blotting, flow cytometry, and fluorescence microscopy. Enzyme-linked immunosorbent assays (ELISAs) often employ enzyme-conjugated antibodies for the sensitive detection of antigens. Biotinylated proteins can be detected using streptavidin-conjugated probes, providing a versatile and robust labeling strategy.

Nucleic Acid Labeling: Bioconjugation enables the labeling of nucleic acids for various applications, including fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In FISH, fluorescently labeled DNA or RNA probes are hybridized to complementary sequences within fixed cells or tissues, allowing for the visualization of specific genetic loci. Labeled nucleotides can be incorporated into PCR products, enabling the detection and quantification of amplified DNA fragments.

Peptide Labeling: Peptides, due to their small size and versatility, are often labeled for use in various assays and imaging studies. Fluorescently labeled peptides can be employed in receptor binding assays, enzyme kinetics studies, and intracellular trafficking investigations. Additionally, labeled peptides are used in drug development to study peptide-receptor interactions and to develop peptide-based therapeutics.

Applications in Biomolecular Imaging

Bioconjugation, when combined with advanced imaging techniques, provides powerful tools for studying biological systems with high resolution and sensitivity. Some key applications include:

Fluorescence Microscopy: Fluorescently labeled biomolecules are extensively used in fluorescence microscopy to visualize the spatial and temporal distribution of targets within cells and tissues. Techniques such as confocal microscopy, total internal reflection fluorescence (TIRF) microscopy, and super-resolution microscopy rely on conjugated fluorescent probes to achieve high-resolution imaging of cellular structures and dynamics.

Magnetic Resonance Imaging (MRI): Bioconjugation is employed to attach contrast agents, such as gadolinium-based compounds, to biomolecules for enhanced MRI imaging. These contrast agents improve the signal-to-noise ratio, allowing for better visualization of anatomical structures and pathological changes. Targeted MRI contrast agents can provide specific imaging of molecular targets, aiding in the diagnosis and monitoring of diseases.

Single-Photon Emission Computed Tomography (SPECT): Radiolabeled probes, such as technetium-99m or iodine-123, are conjugated to biomolecules for use in SPECT imaging. SPECT provides three-dimensional images of the distribution of radiolabeled compounds within the body, allowing for the non-invasive assessment of physiological processes and disease states.

Positron Emission Tomography (PET): PET imaging utilizes positron-emitting radioisotopes, such as fluorine-18 or carbon-11, conjugated to biomolecules for the detection of metabolic and molecular changes in vivo. PET is widely used in oncology, neurology, and cardiology for the diagnosis and monitoring of diseases, as well as in drug development for evaluating pharmacokinetics and pharmacodynamics.

Example of PET (Wei W., et al., 2020)

Future Prospects

The future of bioconjugation in biomolecular labeling and imaging is promising, driven by continuous advancements and expanding application scenarios. Emerging technologies and novel bioconjugation strategies are expected to further enhance the capabilities and versatility of this field.

Advancements in Bioconjugation Chemistry

Ongoing research in bioconjugation chemistry aims to develop more efficient, selective, and bioorthogonal reactions. Innovations such as site-specific bioconjugation, enzyme-mediated bioconjugation, and click-to-release strategies are likely to provide greater control over the labeling process, improving the performance and reliability of conjugates.

Integration with Next-Generation Imaging Techniques

The integration of bioconjugation with cutting-edge imaging techniques, such as super-resolution microscopy, light-sheet microscopy, and multimodal imaging, will enable researchers to visualize biological processes with unprecedented detail and accuracy. These advancements will facilitate the study of complex cellular interactions and dynamic events in real time.

Expansion into New Application Areas

Bioconjugation is poised to expand into new application areas beyond traditional biomolecular labeling and imaging. For instance, the development of targeted therapeutics, such as antibody-drug conjugates (ADCs) and nanoparticle-based delivery systems, relies on precise bioconjugation strategies to ensure selective targeting and controlled release of therapeutic agents.

Personalized Medicine and Clinical Diagnostics

The advent of personalized medicine and the increasing demand for precision diagnostics will drive the adoption of bioconjugation in clinical settings. Conjugated probes and contrast agents tailored to individual patients' molecular profiles will enable more accurate disease diagnosis, monitoring, and treatment, ultimately improving patient outcomes.

In conclusion, bioconjugation has established itself as a fundamental tool in biomolecular labeling and imaging, providing researchers and clinicians with powerful means to study and manipulate biological systems. With ongoing advancements and expanding applications, this technology is set to continue its transformative impact on biomedical research and clinical practice, offering innovative solutions for a wide range of scientific and medical challenges.