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Science/Tech See other Science/Tech Articles Title: Creating A Medical Nanobot - Electrons Regulating Enzymes At Molecular Level On A Chip Medicine's Fantastic Voyage Dear Colleagues, wouldn't it be great if we could get away from all the messy complexity of a cell, even a synthetic cell, and just make the biomolecules we need using high throughput fuidic cells? This field is developing as demonstrated by this research. Gordonov, et al. demonstrate that direct electrical control of a diffusable redox mediator at the surface of a gold electrode in the vicinity of an immobilized enzymatic pathway results in predictable protein oxidation, attenuation of activity, and biochemical signal generation. Their work is summarized by Mahler, the source of Figure 1. Mahler G, Enzyme control on a chip, Nature Nanotechnology, 2014;9:571-572. Gordonov T, Kim E, Cheng Y, et al., Electronic modulation of biochemical signal generation, Nature Nanotechnology, 2014;9:605-610. The gist: Enzymatic cell-free systems are being created in vitro that are not "contained in a bag". "Isolating cellular components and creating a cell-free system allows cell machinery to be engineered for a specific function, while reducing the unwanted interactions that occur within intact cells. Opportunities related to this technology include the ability to synthesize products that are difficult or impossible to produce with other methods; obstacles include the predictable control of these complex biological processes." "One such technology is enzymatic cell-free systems, which could be used to probe metabolic pathways, and synthesize protein therapeutics, small molecules and non-natural products. These in vitro enzymatic systems can generally reach much higher reaction rates than those in vivo, tolerate toxic compounds and function under a wide range of reaction conditions." "One key advantage of in vitro enzymatic systems is the enzyme concentration, which far exceeds cytoplasmic levels. To promote enzyme productivity and pathway control, enzymes can be selectively patterned onto a substrate, which increases their concentration and proximity to the substrate molecules. It also allows for area-based manipulation of one- or multiple-step reaction." A thin layer of chitosan* was deposited on a gold-coated silicon chip. HLPT, a multi-domain fusion protein composed of an N-terminal pentahistidine tag and the bacterial enzymes LuxS and Pfs, was then covalently attached to the chitosan. LuxS and Pfs can synthesize the signal-molecule bacterial autoinducer-2 (AI-2) and homocysteine in a 1:1 ratio from the enzyme pathway precursor, S-adenosyl-homocysteine. HLPT also has a C-terminal pentatyrosine tag that allows it to covalently attach to chitosan's primary amines with the enzyme tyrosinase. *Chitosan is deacylated chitin from crustaceans. See Figure 1. "These experiments measured the conversion of S-adenosyl-homocysteine to homocysteine and AI-2 in the presence of electrically oxidized acetosyringone. Homocysteine synthesis was measured optically or electrochemically, whereas AI-2 was detected biologically with AI-2 fluorescent reporter cells. The results showed that HLPT activity was reduced due to oxidation of its sulfhydryl residues, and that HLPT activity decreased proportionally to the oxidized acetosyringone:HLPT ratio. These results proved that electrically mediated oxidation can control enzyme activity." "They were even able to tune the amount of biochemical product formed by estimating the numbers of electrons needed to deactivate one HLPT molecule for each on-chip reaction and adjusting the charge needed to reach enzymatic activity set-points." This work demonstrates that enzymes can be electrically assembled and tuned on-chip, effectively coupling microelectronic devices and biological function control. Continuing development will be needed to incorporate chitosan-coated chips into microfluidic devices with multiple enzymes (or even an entire biochemical pathway). On-chip biological sensing will be critical in the use of this technology in basic science, drug development, toxicity testing, and bio-protein product manufacturing applications. Personally, I think this microfluidic system will be very complimentary to the synthetic cell systems that are being concurrently developed. The outcome promises the rapid development of clinically useful biomolecules in abundance. I hope we can afford them! Poster Comment: Chip implants to boost production of necessary enzymes? Post Comment Private Reply Ignore Thread
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