NANO 120A

Module 3

Separation of Micro/Nanoparticles viaAC Dielectrophoresis in Different Samples

of Micropipette Tip

Group B – Team 10

Hong Shen ChenA97074642

March 12, 2015

Purpose

In this experiment, particles are separated according to their size and dielectric constant

by an alternating current (AC) dielectrophoretic procedure. A basic setup for the DEP device

consists of a gel filled micropipette tip, and a fluidic chamber adhered to a glass slide. More

importantly, it is necessary to learn how to operate a fluorescent microscope, and to analyze

images because it is the basic tool for all engineers to study materials below several microns. A

quantitative analysis can also be done by using the software Image J, which measures the

intensity of fluoresce. Software AutoCAD and COMSOL are also implemented to simulate the

different geometrical effects of the system, which generates a different gradient of the electric

field.

Background

The separation of micro/nanoparticles for scientific analysis is becoming more and more

important in many fields today, especially in medicine. The ability to isolate micro-particles

from the nano-particles has drawn significant attentions in detecting disease-related DNA

biomarkers for diagnostics and in delivering drug nanoparticles directly in blood for therapies. [2]

Recently, the cancer research has shown a close relationship between cancer cells growing and

the increasing level of cell-free circulating (CFC) DNA along with apoptotic DNA in blood. [2]

However, in order to analyze the DNA fragment of those disease biomarkers, a separation of

high-molecular weight DNA (HMW-DNA), with typical order of 20nm, needs to be separated

first. [2] And for the CFC-DNA, it has an even smaller order than 20nm. Such small particle

requires an even higher conducting condition. [3] Therefore, a unique condition to optimize the

separation of particles needs to be rediscovered; yet, this discovering process can be time

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consuming and pricy. [4] More importantly, AC dielectrophoresis (DEP) is a more efficient

method to separate the disease-related DNA biomarkers from blood cell compared to DC

electrophoresis. DEP has a better capability to separate a variety range of nanoparticles because

it is size dependent, whereas the electrophoresis, experiencing a DC field, is charge dependent.

This is why we do not adopt electrophoresis to separate the disease-related DNA biomarkers

from blood cells. [1][2] DEP is resulted from the movement of the polarized particles due to a non-

uniform electrical field. Some other possible applications of DEP include cell separation, DNA

isolation, bacteria/virus isolation, and manipulation of nanoparticles; yet, most of them are done

under the low conductance convention. [2] Lately, we discover a new DEP method that now

allows nano-scale of HMW-DNA to be separated from blood in a high conductivity environment

using a different geometry of its design. And this is why in our experiment; we are required to

use AutoCad to design a most suitable geometry between electrodes and samples that generate

both high and low field regions to isolate 200 nm fluorescent beads from 10 μm beads. [1] Also,

different solutions are adopted in our experiment to test the effects of a different dielectric

constant on the movement of the nanoparticles. All in all, nano-particles, such as HMW-DNA,

experiences a positive DEP (p-DEP), which forces them to the high-field regions, while the

micron size particles experience a n-DEP force and go to the low-field region. [2][4] To

experience a p-DEP force indicates that the surrounding solution has a less polarizability than the

HMW-DNA, and it is vise versa for blood cells experiencing the n-DEP force under the same

frequency domain. In addition, the polarizability of particles is depending not only on the

solution where it is suspended but also on the frequency of the applied electrical field. [2] In order

to optimize the separation of particles, we need to select a critical crossing frequency, which will

generate a positive sign of Clausius-Mossotti Factor (CMF) for the smaller particles and a

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negative CMF for the \ The DEP force acting on each particle in the AC electric field is

governmenting by three components: particle geometry, Clausius-Mossotti function, and

magnitude of the gradient of the electric field. This can be shown by the following equation, [5]

⟨ FDEP ⟩=2π r3 ε m ℜ [ CM ]∇|E⃑rms|2

for a homogenous spherical particle with a dipole moment; where r is the radius of the particle;

Erms is the root-mean-square of the electric field based on the geometry of the electrodes and the

applied voltage; and ℜ [ CM ] is the real part of the Clausius-Mossotti factor, which is a function

of the permittivity of the particle and the medium, ℜ{ ε p¿−ε m

¿

ε p¿ +2 εm

¿ }, where ε p¿ and ε m

¿, are the

complex permittivities of the particle and the medium, respectively; they can be altered by

changing the frequency, and are defined as, ε¿=ε−i

σω

which shows a inverse relation to the

dielectric constant of the medium. [5] To get the best result or most force on a particle, the Erms

has to be maximized. This can be done by changing the dielectric constant of the medium, or by

utilizing the geometry of the electrodes, to decrease the electric gradient between high and low

field regions. It is also important to note that both dielectric constants and conductivities are

unchanged for a given material, so in order to optimized the CMF component, we need to adjust

the AC frequency to obtain the most separation. All in all, different size of the particles will

require a very different condition accordingly to help achieve the most separation. [2]

Methods

First of all, prepare three different 1 mL solutions in micro-centrifuge tubes for the test;

then mix 5 μL of 10 μm carboxylate beads and 5 μL of 200 nm yellow-green fluorescent beads

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into the solutions of distilled water, 0.1X TBE, and 0.1X PBS. Secondly, prepare a fluidic

chamber by adhering a PDMS well onto a glass slide. (Make sure the gluing surface is clean)

Next, a 5 mL of 2 wt% Agarose gel was prepared in a 20 mL cylinder glass beaker by mixing

~0.1 g of ultra-pure Agarose powder and 5 mL of 10X PBS, and then it was microwaved in 2 to

3 sec at a time, until the solution was completely homogeneous. This can be done by using the

vortex machine. It is also important to make sure there is no precipitation because it can possibly

affect the imaging later. Next, prepare six pipette tips by adding 10 μL of the solution in each

one of them and let them cool and solidify for about a min. The prepared tips were then

submerged in a 50mL beaker with 10mL 10X PBS to keep the gels hydrated. Subsequently,

design a model of the electrode-tip device using AutoCAD (shown in Figure 2) [1] to run the

simulation in COMSOL and to find its predicting particle separating efficiency before testing,

and then attach the fluidic chamber to a microscope using masking tape. Now, begin to prepare

the first sample by adding the solution of distilled water and beads to the fluidic chamber, and

then fill the pipette tips with only 50 μL of distilled water (no beads). It is necessary to ensure

that there is no air bubble showing in the gel when filling the pipette tip. This problem can be

prevent by tapping the sides of it; again the presence of the air bubbles can significantly affect

the clearly when imaging. And then the sample was then positioned at 45° using an arm attached

to the microscope. The bright-field optical microscopy was then adopted to capture a clear image

of the tip in the solution-filled fluidic chamber due to the fluorescence (Shown in figure 1). By

switching the filter to green, and adjusting the exposure, it was possible for us to observe the

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scatter of yellow-green beads. Take a screenshot before any electric field was applied.

Next, insert one short copper wire to top of the tip inside the sample and another one in

the fluidic chamber. The wires were then

connected to a function generator using

masking tape to stabilize the wires. Close

the shutter, and apply an AC electric field

at a 10 kHz frequency, with a potential

difference of 20 Vpp, for 5 min. Notice

that we do not care about which side is

anode or cathode here due to the AC

convention. Turn off the function generator, and then take another screenshot of the tip again

after having applied the electric field. Lastly, remove the tip and capture a final fluorescent

image. Make sure the shutter was closed once again and let the function generator sweep another

frequency from 10MHz to 1Hz in increments, at 20 Vpp. Repeat the same process for two more

times- for the TBE, and PBS solutions. Make sure the fluidic chamber is filled with the proper

solution of the beads accordingly with either TBE or PBS and the tip was filled with the pure

Figure 1: Bright field micrographs taken before on side - 3ms (left) and after AC DEP (right) procedure in

0.8

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Figure 2: Schematic of electrode-tip device designed using AutoCAD software (measurements in mm).

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TBE or PBS solution only. Finally, use the software Image J to analyze the captured

fluorescence images by measuring the average, and maximum brightness.

Reasult

Figures 3 was produced

using COMSOL and

demonstrate the electric

potential gradient of the

system. Figure 3 shows

the high field regions at

each electrode and low

field regions elsewhere.

Arrows are the field

gradient, and it is a

function of the electric

potential.

Figure 3: Electric field gradient displayed as a function of the electric potential and the distance from the electrodes

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Figure 4: Fluorescent micrographs taken before - 40ms (left) and after AC DEP (right) procedure in DI-water solution for 5 min.

Figure 5: Fluorescent micrographs taken before - 40ms (left) and after AC DEP (right) procedure in TBE solution for 5 min.

Figure 6: Fluorescent micrographs taken before - 40 (left) and after AC DEP (right) procedure in PBS solution for 5 min.

Figures 4-6 are micrographs taken before and after the AC DEP procedure was completed.

They show the collection of fluorescent beads on the tip of the device.

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Discussion

After comparing the micrographs before and after of the DEP process under the AC

convention, it is intuitive for us to state that the separation of particles was successful overall.

We were able to separate the green-yellow fluorescent particles from the carboxylate beads in

each sample. Yet, it is important to keep in mind that some samples might perform better than

others. When comparing different solutions based on the change in fluorescence after applying

the AC field, we are able to see that particles separated the quickest and the most in H2O sample,

whereas, in PBS solution, it results as the slowest and the least. The result was expected

according to the equation (FDEP equals to 2 π r3 ε m ℜ [CM ]∇|⃑Erms|2). [5] As the dielectric constant

of the medium decreases, the more conducing it will be; therefore, the salinity of the solution

will increase. As a result, less force will be experienced by particles, making DES harder for

separation. Also based on the same force equation mentioned earlier, it shows the larger particle

will experience more force, therefore separating quicker. By analyzing the micrographs using

Image J, we were able to measure a quantitative fluorescence (brightness) of each sample.

Ideally, both values of max and mean have to have a larger number in each sample after the field

Figure 7: Analyzed results of the intensity from Image J, where 1, 2 for the PBS solution, 3, 4 for the TBE solution, and 5, 6 for the DI-water solution. (Odd – before the electrical field is applied; Even - after)

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is applied, however, according to the figure 7, in water, the mean for the after shows lower. This

contradiction is shown probably because water has the lowest viscosity so it flows fastest. As a

result, it might cleave off some of smaller particles at the tip, and this movement along with the

pushing movement experienced on larger particles can possibly make it happen for water to

experience a lower mean after. Also, theoretically, water should have the greatest max and mean

(most accumulation), than TBE, and than PBS due to the decreasing trend in dielectric constant

of the medium. [5] Yet, in the figure 7, the after max of PBS shows a larger number than the after

max of TBE. This contradiction can be understood by comparing the figure 5 and 6. First, in the

figure 5, we see an overall larger bright area of fluorescence; whereas in the figure 6, the bright

area decreases tremendously. It is important to realize when the bright area we focused are

becoming smaller in PBS sample, the denser the particles are going to be collected due to the

restricting field strength, and therefore the max will show higher because max indicates the

brightest point in image. Brighter points are presented in PBS sample due to more overlapping of

particles. It is also possible that the electrode was inserted to a different depth in each sample

generating a different strength in electrical field, which differs the result from the expected one.

The shorter distance between the electrode and the tip, the stronger field it will generate.

It is also important to sustain the voltage at a Vpp, 20 because an increasing in voltage

would generate more heat on the

electrodes, which could potentially

cause damage. Also, if we altered the

Vpp, a different size of particles can

now be separated. The effect would

also be similar if the frequency were

Figure 8: black line- smaller particle; red line – larger particle; Wcrt – 10kHz (frequency)

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becoming too high; at that point, the circuit starts to act like a DC circuit and to repulse all

particles to the negative low field region. The separation process stops. On the other hand, at

really low frequencies, such as (10 Hz, 1Hz), we were able to observe a super fast pull in and

pull out movement of particles and to experience a large conductance from the experiment.

Therefore, as the conductance increase, the salinity of the solution increase, thus stopping the

reaction.

Conclusion

This experiment was done to gain an understanding of one method that is quite novel and

a valuable technique useful for the separation and collection of micro/nanoparticles for analysis.

Overall, this laboratory experiment served to demonstrate fundamental principles underlying

dielectrophoresis as well as possible biomedical applications for which it could be used. It was

also useful for introducing essential research and engineering software that can be utilized for

designing a device, for better understanding physical properties of a system, as well as for

analyzing images captured using fluorescent microscopy.

Hypothetically, if a $10,000 grant was provided to improve the efficiency of this device,

it would be essential to create highly accurate simulations to better understand problems, such as

the ability to separate even smaller particles (20 nm) or challenges resulting from highly

conductive solutions resulting from a high salinity. It would also be beneficial to optimize the

geometry of the electrode, and the AC potential difference and frequency to determine how some

of these challenges can be overcome. Understanding the essential elements involved for the

creation of the ideal DEP device such as the effect of using different electrodes or coating them

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in another material and experimenting with applying the AC power at different phases to

different electrodes.

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Reference

[1] Song, YJ, Sonnenberg, A, Heaney, Y, Heller, MJ, “Device for dielectrophoretic separation

and collection of nanoparticles and DNA under high conductance conditions”

Electrophoresis (accepted) 2014.

[2] Sonnenberg, A, Marciniak, JY, Krishnan, R, Heller MJ, “Dielectrophoretic Isolation of DNA

and Nanoparticles form Blood”, Electrophoresis V33, 2482-2490, 2012

[3] Krishnan R, Dehlinger DA, Gemmen GJ, Mifflin RL, Esener S and Heller MJ, “Interaction of

nanoparticles at the DEP microelectrode interface under high conductance conditions”,

Electrochemical Communications, V11, #8, 1661-1666, 2009

[4] Krishnan R and Heller MJ, “An AC electrokinetic method for the enhanced detection of

DNA nanoparticles”, J. Biophotonics, V2, #4, pp. 253-261, 2009

[5] Peter R. C. Gascoyne and Jody Vykoukal, “Particle separation by dielectrophoresis”,

Electrophoresis. 2002 July ; 23(13): 1973–1983.

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