Dielectrophoretic force characteristics toward Lactobacillus casei on an oblique-patterned electrode

Dielectrophoresis-based biochips with various microelectrode configurations have been extensively studied within the last five deca des. However, wide-field application is still challenging. This study aims to fabricate an oblique-configuration microelectrode and then utilizes it to determine the dielectrophoretic (DEP) characteristics of Lactobacillus casei based on the generated non-uniform electric field. The electric field distribution on microelectrodes was simulated with Quickfield 6.6 student version. The microelectrodes were fabricated using a copper film on a glass substrate. The DEP characteristic of Lactobacillus casei was investigated by applying a sinusoidal AC signal to microelectrodes in medium solution with an electrical conductivity of 0.05 S/m. The electric-field simulations show that the strongest electric-field was generated on the tip of electrodes spacing, and the weakest electric-field was generated on the inner of electrodes spacing. The negative-DEP force on Lactobacillus casei was observed at the frequency of 60–130 kHz, while the positive-DEP force was observed at the frequency of 380–700 kHz, both at voltage of 2–6 Vpp. The nDEP force led Lactobacillus casei to be pushed toward the weak electric field on the inner of electrodes spacing, and the pDEP force induced Lactobacillus casei to be attracted toward the strong electric-field on the tip of electrodes spacing. This study implies that the proposed oblique-microelectrode platform has promising application for bioparticles separation.


Introduction
Biochip is a miniaturized test device for biosample analysis [1][2][3].Various scientific and engineering concepts are used as the working principles of a biochip system, such as dielectrophoresis, microfluidics, and magnetism [4][5][6].Dielectrophoresis (DEP)-based biochips are very popularly used to identify and analyze biosamples.Dielectrophoresis is a phenomenon that describes the movement of particles due to its polarization in a non-uniform electric field at a microelectrode [7][8].Microelectrodes have various designs or configurations, where each configuration has a specific function [9].One of the Abstract.Dielectrophoresis-based biochips with various microelectrode configurations have been extensively studied within the last five deca des.However, wide-field application is still challenging.This study aims to fabricate an oblique-configuration microelectrode and then utilizes it to determine the dielectrophoretic (DEP) characteristics of Lactobacillus casei based on the generated non-uniform electric field.The electric field distribution on microelectrodes was simulated with Quickfield 6.6 student version.The microelectrodes were fabricated using a copper film on a glass substrate.The DEP characteristic of Lactobacillus casei was investigated by applying a sinusoidal AC signal to microelectrodes in medium solution with an electrical conductivity of 0.05 S/m.The electric-field simulations show that the strongest electric-field was generated on the tip of electrodes spacing, and the weakest electric-field was generated on the inner of electrodes spacing.The negative-DEP force on Lactobacillus casei was observed at the frequency of 60-130 kHz, while the positive-DEP force was observed at the frequency of 380-700 kHz, both at voltage of 2-6 Vpp.The nDEP force led Lactobacillus casei to be pushed toward the weak electric field on the inner of electrodes spacing, and the pDEP force induced Lactobacillus casei to be attracted toward the strong electric-field on the tip of electrodes spacing.This study implies that the proposed oblique-microelectrode platform has promising application for bioparticles separation.microelectrode configurations used for biosamples manipulation is an oblique configuration [10].DEPbased oblique microelectrodes can be used to focus and separate biosamples due to polarization, which causes bioparticles to be attracted towards the electrode with a strong electric field (positive-DEP force) and pushed towards the electrode with a weak electric field (negative-DEP force) [7,9].The DEP force can be expressed using equation (1).
The DEP force is influenced by the particle radius (), vacuum permittivity (Ɛ 0 ), relative permittivity of the suspension medium (Ɛ  ), the real part of the Clausius-Mossitti () factor (  (  )), and the electric field gradient (∇).The Clausius-Mossotti factor shows the degree of particle polarization and can be written in the form of equation (2).
Where Ɛ  * = Ɛ  − σ p ω ⁄ or Ɛ  * = Ɛ  − σ m ω ⁄ with Ɛ  is the permittivity of the particle, Ɛ  is the permittivity of the medium, σ  is the electrical conductivity of particle, σ  is the electrical conductivity of suspension medium,  is an imaginary number with  = √−1, and ω is angular frequency.The appropriate setting of each parameter is necessary to carry out biosample manipulation such as Lactobacillus casei.
Lactobacillus casei is a microbiota living in the human body and has been widely used as a probiotic because of its ability to kill disease-causing bacteria and help maintain the immune and healthy digestive systems.In its use, isolation, separation, sorting, and various other manipulations are required.In this study, an oblique microelectrode array was used to determine the dielectrophoresis force characteristics of Lactobacillus casei.DEP characteristics of a bioparticle provide a promising opportunity for bioparticle separation, sorting, isolation, and other manipulations.

Lactobacillus casei preparation
Lactobacillus casei samples were suspended in a medium consisting of water, sucrose, skim milk powder (3.4%), dextrose, and synthetic flavors.The samples were then diluted using sterile distilled water with a ratio of 1:9 and an electrical conductivity of the samples in medium was set at 0.05 S/m.

Simulation of electric field distribution on microelectrodes
The electric field distribution of the oblique microelectrode pattern was simulated using Quickfield 6.6 student version software.Simulation was carried out to determine distribution of the non-uniform electric field.The simulation began by "drawing" the geometry of the electrodes as shown in Figure 1a.Then the electrical permittivity and voltage parameters were defined.Electrical permittivity was defined as the relative electrical permittivity of air which has a value of 1, while the voltage was applied to both electrode terminals as the positive pole (U+) with the voltage of +5 V and as the negative pole (U-) with the voltage of -5 V (Figure 1b and 1c ).The next stage was "meshing" to divide the plane of the oblique geometry into small elements (Figure 1d).The final stage was "solving" to provide a solution in the form of electric field distribution based on the geometry and voltage applied.The simulation results of the electric field distribution were then compared to theoretical studies.

Design and fabrication of microelectroda and equipment set up for DEP test
Microelectrodes of copper films on glass substrates were fabricated using pattern transfer techniques.Each microelectrode has a terminal with a length of 1200 µm and a width of 5000 µm, as well as an oblique electrode with a length of 3500 µm, a width of 500 µm, and an inner electrode spacing of 200 µm.Each design varied the electrode tip spacing, namely 200 µm, 150 µm, and 100 µm.The oblique pattern design is as shown in Figure 2a.The microelectrode fabrication process was employed in several stages.The first stage was the microelectrode pattern transfer process.The copper film with a thickness of 0.06 mm was cut to the size of the glass substrate and attached then to the glass substrate which has a size of 25x76 mm and a thickness of 1 mm.The microelectrode pattern was then transferred to copper film using oil-based lotion.The lotion contains an active substance, which can transfer the toner ink pattern from paper to copper film.The second stage was the etching process which was carried out using a 0.1 M FeCl 3 solution.The microelectrode was immersed in the FeCl 3 solution while being gently agitated so that the unnecessary copper film layer was removed from the surface of the glass substrate.Next, the etched microelectrodes were cleaned with water and acetone.The final stage in microelectrode fabrication was wiring the electrode terminals, as shown in Figure 2b.The equipment for the experiment was set up as in Figure 2c, which consists of microelectrodes, a microscope with a CCD camera, a function generator, and a computer.The microelectrode was connected to a function generator whose magnitude of a sinusoidal AC voltage (Vpp) and frequency were regulated.The DEP force phenomenon was observed using a microscope displayed on a computer screen.

Results of lactobacillus casei sample preparation
Lactobacillus casei was used as a bioparticle test sample to identify the DEP phenomenon on the fabricated microelectrode.Lactobacillus casei samples were prepared in a medium with an electrical conductivity of 0.05 S/m.Observation of the shape using an optical microscope and validation of sample size were carried out using ImageJ software after obtaining the image from the microscope, as shown in Figure 3.The shape of Lactobacillus casei observed was rod-shaped, with a length of 3.28 ± 0.55 µm and a width of 1.04 ± 0.20 µm which corresponds to the reference (Table 1).

Simulation results of electric field distribution on oblique microelectrodes
Figure 4 shows the simulation results of the electric field distribution.The strongest electric field was identified in the space between the electrode tips, which is marked with a red contour, and the weakest electric field occured in the inner space between the electrodes, which is marked with a purple contour.These show that the oblique microelectrode pattern exhibited a gradient in electric field strength or a non-uniform electric field, which allows the DEP phenomenon to occur.

Testing of DEP characteristics of lactobacillus casei
Testing of the characteristics of DEP of Lactobacillus casei was conducted at voltage variations, namely 2 Vpp, 4 Vpp, and 6 Vpp.Higher voltages could not be applied to the testing process because it could cause bubbles to appear.Application of low or very high frequencies could also cause bubbles to form.
Bubbles was formed at low frequencies between 10-50 kHz and high frequencies >700 kHz.These bubbles can disrupt DEP observations because they affect the movement of Lactobacillus casei.Based on observations, there was a difference in the movement of Lactobacillus casei before and after the sinusoidal AC voltage was applied.Before the voltage was applied, Lactobacillus casei would move randomly, while after the voltage was applied, Lactobacillus casei would move from the inlet to the outlet following the strong or weak electric field regions.The results show that that nDEP was observed at a frequency of 60-130 kHz while pDEP was obtained at a frequency of 380-700 kHz, when AC voltage of 2-6 Vpp was applied.Figure 5 shows an illustration of the DEP phenomenon of Lactobacillus casei observed during testing.Figure 5a shows the negative dielectrophoresis phenomenon (nDEP) where Lactobacillus casei moved towards the weak electric field area, and Figure 5b  The nDEP phenomenon observed on the microelectrode is as shown in Figure 6 and the pDEP phenomenon is shown in Figure 7. Lactobacillus casei could not be completely attracted to the tip of the oblique electrode due to the large space between the electrode tips.This is in accordance with theories explaining that Lactobacillus casei can be attracted to strong field areas, whereas in the space between electrodes Lactobacillus casei cannot be attracted to strong field areas due to the relatively smaller DEP force in the space for size of Lactobacillus casei.The red arrows in the image denote the direction of movement of Lactobacillus casei.The dielectrophoresis curve in Figure 8 shows that the pDEP and nDEP characteristics of Lactobacillus casei can be obtained by adjusting the cross-over frequency (f co ) at varying spacing between electrode tips (Figure 8a) and at varying voltage applied (Figure 8b).As an implication, setting the electrical signal frequency beyond the f co window can distinct the movement of Lactobacillus casei to a certain direction zone.In this case, the direction zones are on the inner of electrodes spacing and on the tip of electrodes spacing.These phenomena can be employed for separation of Lactobacillus casei by controlling electrical operating parameters of electrode such as frequency and voltage peak-to-peak (Vpp).Each bioparticle has a unique DEP characteristics, therefore this platform can also be applied for others bioparticles manipulation.

Conclusions
The results of experiment reveal the dielectrophoresis force characteristics of Lactobacillus casei on an oblique-patterned microelectrode.The oblique-microelectrodes show the non-uniform electric field allowing dielectrophoresis to occur where the strong electric field areas are in the spaces between the electrode tips, while the weak field areas are in the inner of electrode spaces.DEP characteristics of Lactobacillus casei show that overall nDEP begins to be observed at a frequency of 60-130 kHz, while pDEP is observed at a frequency of 380-700 kHz, with an operating voltage of 2-6 Vpp.The results of this research show the potential application for manipulating bioparticles using oblique-configuration microelectrodes which can be used for the isolation, separation, and sorting of bioparticles.

Figure 1 .
Figure 1.Procedures for simulation of electric field distribution, (a) Drawing of an oblique microelectrode pattern, (b) Parameter setting of medium, (c) Parameter setting of relative electric permittivity, and (d) Meshing (a) (b) (c) (d)

Figure 2 .
(a) Design of oblique microelectrode pattern, (b) Results of microelectrode fabrication and wiring, (c) Set up of experimental equipment for DEP test.

Figure 6 .Figure 7 .
Figure 6.Lactobacillus casei was pushed into the space between the electrodes in the weak field area (nDEP), at a voltage of 2-6 Vpp and a frequency of 60-130 kHz

Figure 8 .
Figure 8.The pDEP dan nDEP curves of Lactobacillus casei on (a) Variation of spacing between electrode tips, (b) Variation of applied voltage.

Table 1 .
Lactobacillus casei measurement validation results