Bio-orthogonal Crosslinking
Bio-orthogonal Crosslinking - Related Videos
03:47
Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay
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2025
This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.
14:02
Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
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Cited by 5 •
2018
A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.
10:54
Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry
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Cited by 7 •
2017
Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.
11:15
Nanosponge Tunability in Size and Crosslinking Density
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Cited by 1 •
2017
This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.
09:06
Preparation of DNA-crosslinked Polyacrylamide Hydrogels
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Cited by 5 •
2014
Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.
FAQs
The key reaction pairs tetrazines with strained alkenes in a rapid, catalyst-free click process. Because these complementary groups react selectively in biological environments, crosslinking can proceed under mild, aqueous conditions rather than requiring conditions that disrupt native cellular chemistry. This selectivity supports formation of biomaterial networks in the presence of living cells.
Once formed, the network's properties can be tuned through the biomaterial components and their crosslinking behavior. The resulting structure may provide selected mechanical properties, degradation characteristics, and biochemical presentation. In bioengineering, these parameters matter because they influence how a material supports cells, presents signaling molecules, or releases therapeutic cargo.
Avoiding an added catalyst helps keep the reaction compatible with biological environments and native cellular chemistry. The tetrazine-strained alkene pairing can therefore be used under mild, aqueous conditions, where functionalized polymers or proteins are joined without introducing a catalyst-dependent crosslinking step. This feature is especially relevant when cells must remain present during material formation.
A basic workflow begins with functionalizing polymers or proteins with complementary reactive groups. The modified components are then brought together in a mild, aqueous environment, where the groups react and create a covalent network. Depending on the design, this process can be performed while cells are present, enabling fabrication of cell-containing biomaterials.
Injectable hydrogels are a major format because crosslinking can create the network after delivery at the intended site. The same strategy can immobilize signaling molecules, encapsulate cells, support controlled drug delivery, or contribute to tissue repair. These uses connect reaction selectivity with practical biomaterial functions in regenerative medicine and other biomedical applications.
The ability to crosslink in the presence of living cells enables minimally invasive biomaterial fabrication, a useful feature for regenerative medicine. Engineers can create cell-containing hydrogels and related materials while maintaining compatibility with cellular chemistry. This capability expands applications toward tissue repair, biochemical signaling, and controlled delivery while allowing the network's properties to be adjusted for biological needs.