Difference between revisions of "Microoxen: Microorganisms to Move Microscale Loads."

From Soft-Matter
Jump to: navigation, search
Line 22: Line 22:
 
Figure 1 also shows the chemistry used to attach polystyrene beads to the surface of the algal cells.  After coating the beads with peptide, cells and beads were mixed together.  Random collision between a bead and cell resulted in the two being bound together.  Since the binding of beads to cells is not targeted, the beads could end up at any point on the algae's outer membrane and it is possible for multiple beads to be stuck to one algae.  Since these binding events are independent of one another, we expect the distribution of number of beads per cell to be Poisson distributed and controlled by the average number of beads per cell.[[http://en.wikipedia.org/wiki/Poisson_distribution]]
 
Figure 1 also shows the chemistry used to attach polystyrene beads to the surface of the algal cells.  After coating the beads with peptide, cells and beads were mixed together.  Random collision between a bead and cell resulted in the two being bound together.  Since the binding of beads to cells is not targeted, the beads could end up at any point on the algae's outer membrane and it is possible for multiple beads to be stuck to one algae.  Since these binding events are independent of one another, we expect the distribution of number of beads per cell to be Poisson distributed and controlled by the average number of beads per cell.[[http://en.wikipedia.org/wiki/Poisson_distribution]]
  
After the beads are attached to cells, the cells are introduced into a PDMS microfluidic channel.  The channel has four PDMS walls that have been passivated by flowing a 5% BSA in PBS buffer solution through the channel just after bonding via the standard oxygen plasma treatment.  The coating of BSA on the walls minimizes adhesion of the algae to the PDMS and allows experiments to be run for 8 or more hours.  Figure 2 shows a schematic of the channel as well as an image sequence of swimming algae.  The cartoon circles indicate which of two LEDs is switch on to an intermediate intensity.  The cells exhibit positive phototaxis and swim toward the light that is on.  The dotted line circle indicates a cell carrying a bead, while the dotted line square indicates a cell without a bead.  The two remain roughly a constant distance apart, indicating that the bead does little to impede the swimming of the algal cell it is attached to.
+
After the beads are attached to cells, the cells are introduced into a PDMS microfluidic channel.  The channel has four PDMS walls that have been passivated by flowing a 5% BSA in PBS buffer solution through the channel just after bonding via the standard oxygen plasma treatment.  The coating of BSA on the walls minimizes adhesion of the algae to the PDMS and allows experiments to be run for 8 or more hours.  Figure 2 shows a schematic of the channel as well as an image sequence of swimming algae.  The cartoon circles indicate which of two LEDs is switch on to an intermediate intensity.  The cells exhibit positive phototaxis and swim toward the light that is on.  The dotted line circle indicates a cell carrying a bead, while the dotted line square indicates a cell without a bead.  The two remain roughly a constant distance apart, indicating that the bead does little to impede the swimming of the algal cell it is attached to.  Cell carrying beads were routinely able to swim at an average speed of 100 microns/second for total distances of 16-20cm before colliding with and adhering to other cells, beads, or debris in the microchannel.
  
 
Figure 3 shows how payloads are delivered to their destination.  Once the bead bearing algae have arrived at their final destination, a UV light (365nm) is focused on them through a 20x microscope objective.  From test experiments, illumination with intense UV light for 5-15 minutes had little effect on cell viability.  The amount of time required to photocleave the beads from the algae varies, but is on the order of 10s of seconds.  For the image sequence shown, the cell was illuminated with UV light for 20 seconds before the first frame.  After 18 additional seconds (frames B-J), the bead has detached from the cell and started to diffuse away.  What is unclear from the figure and paper text is if the detached bead was specifically targeted and what the spot size is for the UV illumination.
 
Figure 3 shows how payloads are delivered to their destination.  Once the bead bearing algae have arrived at their final destination, a UV light (365nm) is focused on them through a 20x microscope objective.  From test experiments, illumination with intense UV light for 5-15 minutes had little effect on cell viability.  The amount of time required to photocleave the beads from the algae varies, but is on the order of 10s of seconds.  For the image sequence shown, the cell was illuminated with UV light for 20 seconds before the first frame.  After 18 additional seconds (frames B-J), the bead has detached from the cell and started to diffuse away.  What is unclear from the figure and paper text is if the detached bead was specifically targeted and what the spot size is for the UV illumination.

Revision as of 14:12, 22 April 2009

"Microoxen: Microorganisms to Move Microscale Loads"
Douglas B. Weibel, Piotr Garstecki, Declan Ryan, Willow R. DiLuzio, Michael Mayer, Jennifer E. Seto, & George M. Whitesides
PNAS 102(34) 11963-11967 (2005)


Soft Matter Keywords

algae, phototaxis, photochemistry, beast of burden

Figure 1. Transport system used in this experiment. (A) Power (1-7) and recovery (8-11) strokes of algae. (B) Structure of the peptide used to attach beads to cells. (C) Reaction used to produce peptide-coated beads. (D) Micrograph of bead attached to algal cell. The bead is attached to the cell slightly above the current focal plane and so appears slightly out of focus.
Figure 2. (A)&(B) Schematics of LED/microfluidic channels used to steer the algae. (C) Image of bead attached to algal cell. (D)-(O) Series of frames showing a cell carrying a bead being steered back and forth in the microfluidic channel using positive phototaxis (cell is attracted to the LED that is on).
Figure 3. (A) Photo reaction that will cleave beads from algal cells. (B)-(M) Time series showing release of a bead from a cell carrying two beads. The cell was illuminated with UV light for 20 seconds before frame (B) and the time between frames is 2 seconds.

Summary

The authors detail a very novel approach to transporting small payloads using biological motors. Instead of attaching synthesized motors to a load, Weibel, et al. attach the load to a microorganism, aka a microscale beast of burden. This allows them to steer the transport of the load by controlling the locomotion of the organism. In this case, the unicellular photosynthetic algae Chlamydomonas reinhardtii was chosen for its robust locomotion, phototactic characteristics, and ease of culture. The simulated loads in these experiments were surface modified polystyrene beads. The peptide used to attach the bead to the algal cell contained a photo-active group that allows the bead to be cleaved from the cell when exposed to UV light of the appropriate wavelength. In this way, the beads can be delivered to a particular location and then released.

Practical Application of Research

This system works nicely for transporting microscale objects over relatively long distances (10s of centimeters). As Weibel, et al. point out, this system cannot be scaled down to the nanoscale, but does have the advantage of using an existing organism, which precludes engineering the control of biological motors attached directly to loads. One challenge that must be addressed before this is a fully viable system is controlled attachment of beads to the cells. An ideal system would allow precise placement of the bead on the cell surface so it doesn't impede locomotion.

Moving Loads with Tiny Oxen

Chlamydomonas reinhardtii (CR) is a type of photosynthetic algae that propels itself using two flagella. The flagella are approximates 12 microns in length and execute a breaststroke-like motion, as shown in Figure 1. The flagella beat at a frequency around 40-60 Hz and can propel the 10 micron diameter algae at velocities in the neighborhood of 100-200 microns/second. As the cells swim, they rotate counterclockwise above their longitudinal axis, tracing out a helical path. CR cells exhibit phototactic ability, with a maximum response at 505nm and a secondary response at 443nm. At high intensities, the cells exhibit negative phototaxis, swimming away from the light source, while at intermediate intensities, the cells are attracted to the light (positive phototaxis). Weibel, et al. find that attaching a polystyrene bead (1-6 microns in diameter) to the cell has little effect on the algae's locomotion. Only when the bead was attached near the flagella or the algae were swimming in confined channels such that the bead occasionally made contact with the channel walls, did the trajectory and velocity vary significantly from that of a cell swimming without a bead attached.

Figure 1 also shows the chemistry used to attach polystyrene beads to the surface of the algal cells. After coating the beads with peptide, cells and beads were mixed together. Random collision between a bead and cell resulted in the two being bound together. Since the binding of beads to cells is not targeted, the beads could end up at any point on the algae's outer membrane and it is possible for multiple beads to be stuck to one algae. Since these binding events are independent of one another, we expect the distribution of number of beads per cell to be Poisson distributed and controlled by the average number of beads per cell.[[1]]

After the beads are attached to cells, the cells are introduced into a PDMS microfluidic channel. The channel has four PDMS walls that have been passivated by flowing a 5% BSA in PBS buffer solution through the channel just after bonding via the standard oxygen plasma treatment. The coating of BSA on the walls minimizes adhesion of the algae to the PDMS and allows experiments to be run for 8 or more hours. Figure 2 shows a schematic of the channel as well as an image sequence of swimming algae. The cartoon circles indicate which of two LEDs is switch on to an intermediate intensity. The cells exhibit positive phototaxis and swim toward the light that is on. The dotted line circle indicates a cell carrying a bead, while the dotted line square indicates a cell without a bead. The two remain roughly a constant distance apart, indicating that the bead does little to impede the swimming of the algal cell it is attached to. Cell carrying beads were routinely able to swim at an average speed of 100 microns/second for total distances of 16-20cm before colliding with and adhering to other cells, beads, or debris in the microchannel.

Figure 3 shows how payloads are delivered to their destination. Once the bead bearing algae have arrived at their final destination, a UV light (365nm) is focused on them through a 20x microscope objective. From test experiments, illumination with intense UV light for 5-15 minutes had little effect on cell viability. The amount of time required to photocleave the beads from the algae varies, but is on the order of 10s of seconds. For the image sequence shown, the cell was illuminated with UV light for 20 seconds before the first frame. After 18 additional seconds (frames B-J), the bead has detached from the cell and started to diffuse away. What is unclear from the figure and paper text is if the detached bead was specifically targeted and what the spot size is for the UV illumination.


written by Donald Aubrecht