Paramagnetic Bead Method

For this method paramagnetic beads made from iron-oxide are coated in antibodies that will bond to cells. By placing a permanent magnet on top of a very fine needle (insect pins/ dissecting needles) with tip size ranging from 4-10micron in diameter, we will use magnetic fields to remove the beads from a well. Beads act as antibodies, therefore if a bead is picked up then a cell would be picked up as well. After much frustration and many tests, it was ultimately found that this method could successfully remove magnetic beads from micro array wells. Elaborated below is the Testing Setup Evolution, Testing, and Video of Testing.

Table 1. Pros and Cons of Paramagnetic Bead Method

Testing Setup Evolution:

 Figure 1. Neodymium magnet (red highlight), needle (white highlight),

                                        and micro-array slide (yellow highlight)                             

Figure 2. Testing setup showing the camera focused on the needle tip.  

Note that the Z axis is secured to a stand but not to the table.

Figure 3. Z axis firmly mounted to microscope where two cameras circled in blue

are focused on the needle (one from below one obliquely pointing down

on the right). On the left is a light source circled in red.                         

Figure 4. Final testing setup. A much stronger magnet force was needed than expected

Testing:

It was seen that when using 5 of the small neodimium magnets the iron oxide beads floating in water would be attracted to the magnetized needle. The video below shows a magnetized needle placed in a heavily diluted iron oxide bead solution; filming began after the needle had been in the solution for about 1 minute and it can be seen that particles are still attracted to the needle.

The first test showed that a needle could extend the magnetic field to a tip because the beads floating in water were very attracted to the needle. Next, it was necessary to determine the best dilution ratio of beads to water such that a bead could be singled out and extracted. Ratios of 2:1, 5:1, and 20:1 were all tested and it was found that the 20:1 ratio offered the best bead distribution.

Figure 5. From left to right: 2:1 ratio, two parts water to one part bead solution; 5:1 ratio, five parts water to one part bead solution; 20:1 ratio, twenty parts water to one part bead solution.

After the bead distribution ratio was solved, it was time to test the extraction of beads from wells. To test bead extraction, the method was first broken into three parameters: needles, magnetism, and trajectory. A variety of different needles were purchased with different lengths and diameters. The needles were all made out of carbon steel that could be magnetized and some of the needles had black oxide coatings. Figure 6 shows the different needles tested where the silver needles are stainless steel and the black needles are black oxide coated stainless steel. It was found that the larger the contact area was between the needle end and the magnet, the stronger the magnetic field could be at the needle tip. Also, if the needle was shorter the field could be stronger at the tip. When the needle was too short the magnets would block the light source and make testing impossible to see but 38mm was found to be a successful length.

Figure 6. The variety of different needles that were purchased to test the needle parameter. The red circle indicates the needles that worked best for cell extraction.

Originally the 40 circle magnets were purchased but it was seen through testing that much more magnetic force would be necessary for extraction. To supplement the original magnet purchase, stronger magnets were purchased. By achieving too much magnetism at the tip of the needle it was easy to remove magnets until the forces at the tip were strong enough but not too strong. From left to right, the holding force of each magnet was 2 lbs, 9.8 lbs, 15.7 lbs, 17.4 lbs, 24.5 lbs.

Figure 6. The variety of magnets that were required to test cell extraction

The three trajectories tested were: poking the micro-array until deformation of the array could be qualitatively observed, not touching the micro-array, and sweeping the needle over the micro-array in the area where pickup was desired.

Figure 7. From left to right: array being poked, needle not touching array, and needle sweeping over array.

Many testing iterations were completed by varying the three parameters until a good combination was observed to successfully extract beads. The table below shows all of the testing parameters as well as the quantity of times each parameter was tested and the result of each test.

Table 2. Results from Testing Paramagnetic Bead Method with Different Parameters

Video of Testing:

Video of a successful pick:

In the video it can be seen that a bead is magneticaly attracted to the tip of the needle at around :05 seconds.

Video of an unsuccessful pick:

In the video it can be seen that beads are actually placed onto the array at around :18 seconds. The reason beads were placed was because the magnetic strength at the end of the tip was not strong enough. This was why so many magnets were required for the final design to successfully extract beads from wells.