We have successfully demonstrated the assembly of 5 Bytes within one of our microfluidic chips. The construct was made up of the anchor and 5 Bytes (2 genes that alternated). Each Byte chamber held 4.2 uL of their Byte along with ligase and ligase buffer (totalling 5 uL in each chamber). This is a very small amount compared with the volumes used in the off-chip protocol. The total mass of the magnetic beads was 8 micrograms which is 2 uL of the original bead solution. The procedure described in the previous section was carried out. The beads were left in each chamber for 15 minutes. The total time was less than 2 hours and was executed at room temperature. After digesting the construct off of the bead with the USER enzyme, the construct was ran on a gel. The gel showed 5 very dim but distinct bands (which is expected with the low reagent volumes). The contents of the chambers were also run on the same gel to verify that there was no contamination from other Byte chambers. The size of the construct implied by the bands indicate that the microfluidic assembly process was a success! Repeated experiments verified these results.

The XY stage in the robot was built by a few undergraduates here at the U of A as their capstone design project. This XY stage is what moved the magnet around which controlled where the beads went on the microfluidic chip. Since then, this XY stage has been incorporated into another machine meant to combine many lab-on-a-chip functions into one device including PCR and CE for sequencing. That is why the machine is so big; the internal laser and camera take up a lot of space. The software can be controlled from a Mac or PC. Since the chip's channels were curved, it was somewhat cumbersome to attain the XY positions that the magnet needed to go. But once the positions were known, the rest was easy.

Figure 3.

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?

Figure 4.

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 are 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. Since there was limited space, curving the channels is all we could do to increase channel length. 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 suggests that the Laplace flow from the washing chamber is enough to cleanse the beads of DNA that has not ligated.

Many of the procedures needed for Byte assembly have been accomplished on a microfluidic chip in labs across the world. Although not used for Byte assembly and not accomplished on the same chip, these on-chip protocols have shown that PCR, purification, digestion, electromagnetic bead manipulation, ligation, cell separation, and electroporation (transformation) can all be achieved on-chip. The future will bring many of these procedures together onto one chip allowing the researcher to choose their artificial plasmid on the computer, and upload the program to the microfluidic chip's microprocessor.

Figure 5.

The diagrams show what a Biofab-on-a-chip might look like. It would be a large scale integration of previously independent lab-on-a-chip procedures. Starting on the left of the first labeled diagram:

When the Bytes are ready in their chambers, the supply of beads is split into groups by the external magnet or the internal micro-electromagnets. Each portion is transferred to a different Byte chamber and attaches a Byte to the anchor. After ligation is done, each portion of beads is led to another chamber (through the wash canal) where each bead portion attaches a second Byte. The process continues until a certain amount of Bytes is on each bead.

Figure 6.