Using digital microfluidics, recombinant enzyme technology, and magnetic nanoparticles we have created a functional prototype of an artificial Golgi organelle. Analogous to the natural Golgi, which is responsible for the enzymatic modification of glycosaminoglycans immobilized on proteins, this artificial Golgi enzymatically modifies glycosaminoglycans immobilized on magnetic nanoparticles. Specifically, in the prototype phase we have successfully immobilized heparan sulfate onto magnetic nanoparticles and enzymatically modified the glycosaminoglycan using 3-O-sulfotransferase in a droplet based digital microfluidic device. After enzymatic treatment, the immobilized heparan sulfate showed high affinity for fluorescent antithrombin III, confirming its successful modification by 3-O-sulfotransferase.
Work is currently underway to further develop the artificial Golgi into a nano-scale lab-on-a-chip device for the synthesis and screening of specific glycans. This will be accomplished with the expansion of the chip to include an artificial endoplasmic reticulum to incorporate the synthesis of the core protein so the entire glycosylation pathway can take place. The combination device will provide a platform for the high throughput synthesis of small amounts of glycosaminoglycans for biological and pharmacological evaluation. In addition, this tool may be used in other processes such as the design of biosynthetic heparin, which could replace current unsafe methods of heparin production from animal tissue, cell typing, assays, and toxicology testing.
Collaborator: Prof. R.J. Linhardt
Figure 1. An artificial Golgi and ER are inspired from their natural counterparts. (a) An illustration of the Golgi and ER organelles of a eukaryotic cell. Proteins are synthesized in the ER, and then translocated to the Golgi for posttranslational modification. Modified from Xu et al.1 (b) The design of an artificial Golgi/ER digital microfluidics chip for the biosynthesis of the HS is shown. HS core protein is first synthesized and immobilized on a magnetic nanoparticle in the artificial ER. The particle holding core protein is then transferred into the artificial Golgi portion of the digital microfluidics chip where it is glycosylated to yield HS. The large boxes (multicolor) are reagents and enzyme reservoir electrodes, and the small boxes (light green) are electrodes for droplet movement, mixing, and sequestration. Modified from Martin et al.2 (c) A depiction the process of in vitro transcription/translation and glycosylation in the artificial ER/Golgi device. Figure taken from Martin et al.3
- Xu, D., and Esko, J.D. A Golgi-on-a-chip for glycan synthesis. Nat. Chem. Biol. 2009, 5, 612-613.
- J.G. Martin, M. Gupta, Y. Xu, S. Akella, J. Liu, J.S. Dordick, and R.J. Linhardt (2009), "Toward an Artificial Golgi: Redesigning the Biological Activities of Heparan Sulfate on a Digital Microfluidic Chip", J. Am. Chem. Soc. 131, 11041-11048.
- J.G. Martin, J.M. Beaudet, J.S. Dordick, and R.J. Linhardt (2010), "Artificial Organelles: Digital Microfluidic Platform for Proteoglycan and Glycoprotein Biosynthesis", The Scientific World Journal 10, 997-1000.