Team:Alberta/Project/Microfluidics

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University of Alberta - BioBytes










































































































Lab-on-a-Chip BioByte Assembly

Microfluidic chips bring several advantages to the table. These so called Lab-on-a-Chip devices require only small amounts of reagents. They are portable, inexpensive, typically faster, more efficient, and able to be automated. We have built and tested a prototype chip to execute our novel [[BioByte assembly method]] (4 of them are pictured above). With these chips, we have successfully demonstrated the assembly of 5 genes using 20 times less reaction volumes than what was used off-chip.

How The Chip Works

Many design configurations were considered. For the proof of concept, the simplest design was chosen; it contains no valves or pumps.

The chip consists of a central chamber surrounded by and connected to 8 outer chambers through micro-channels. No two outer chambers are directly connected. The washing chamber is in the middle because it needs to be accessed after every BioByte addition. To assemble the BioBytes, all of the chambers are filled with their appropriate solutions (see diagram above). The beads are dragged with an external magnet from their chamber into the washing chamber, then into the Anchor chamber. After a short period, the Anchor is attached to the bead and the bead can now be dragged out of the Anchor chamber and into the washing chamber. The beads can then be moved into the 1st BioByte chamber. After a short time, the 1st BioByte is ligated to the Anchor on the bead. The beads are then moved to the next BioByte chamber where the next BioByte is ligated. The beads keep on moving from chamber to chamber in this fashion until all of the BioBytes are assembled into one construct. After assembly is done, the beads are recovered and digested to release the construct from the bead. The construct is then ready for transformation.

FAQs

Q. What stops the DNA in the outer wells from diffusing into the washing chamber and contaminating all of the other chambers?

A. We set up Laplace flow originating from the central washing chamber.

Q. What is Laplace flow?

A. Laplace flow is the term used for the flow of liquid caused by differences in pressure. So by filling the central washing chamber to a height slightly higher than the outer chambers, a slow and steady flow against the concentration gradient of the DNA was accomplished.

Q. How do you stop the chamber contents from evaporating?

A. We normally cap the chambers with a small volume of oil. Without the oil, the small reaction volumes can evaporate in less than 5 minutes.

Q. What are some of the physical characteristics of the microfluidic chip?

A. The chip is made of 2 glass layers. The micro-channels are etched onto the top of the bottom layer, and the chambers are part of the top layer. The channels are 45 micrometers by 100 micrometers. The chip itself is approximately 1 inch by 3 inches. Each outer chamber can hold 5 uL of liquid. The central washing chamber volume varied from chip to chip ranging from ~2 uL to 20 uL. Some chips had a closed central washing chamber, but were no longer used after unintentional Laplace flow was found to be largely uncontrollable. The chips were easily reusable after a quick wash.

Q. How do you fill the channels?

A. Once liquid touches the central washing chamber, capillary flow rapidly fills the channels. The outer chambers are then ready to be filled.

Q. Why were the channels made to curve like they do? Why are they not simply coming straight out of the washing chamber towards the outer chambers?

A. The first reason was to increase the length that the DNA had to travel if diffusion turned out to be a problem. The second reason was so that we could visually confirm that Laplace flow was working by filling the outer chambers with orange dye, and the central washing chamber with blue dye. Shorter channel lengths would have made this hard to see.

Q. How do you know that the beads do not drag unwanted DNA around to contaminate other chambers?

A. In our tests, we've recovered the contents of the chambers to run them on a gel and see if any DNA from a different chamber has entered. No measurable amount of DNA was found to contaminate other chambers (including the wash chamber). This seems to suggest that the Laplace flow from the washing chamber is enough to cleanse the beads of DNA that has not ligated.

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