Sheathless Dielectrophoresis-Based Microfluidic Chip for Label-Free Bio-Particle Focusing and Separation ^†

Abstract

This paper presents a novel microfluidic dielectrophoresis (DEP) system to focus and separate cells of similar size based on their structural differences, which is more challenging than separation by size. Because, in this case, the DEP force is only proportional to the polarizabilities of cells, we used live and dead yeast cells as bio-particles to investigate the chip efficiency. Our designed chip consists of three sections. First, we focused on cells at the center of the microchannel by employing a negative DEP phenomenon. After that, cells were separated due to the different deflection from high-electric-field areas. Finally, a novel outlet design was utilized to facilitate separation by increasing the gap between the two groups of cells. The proposed sheath-free design has one inlet for target cell injection requiring only one pump to control the flow rate, which reduces costs and complexity. Successful discrimination of the particles was achieved by using DEP force as a label-free and highly efficient technique. As an accessible and cost-effective method, soft lithography with a 3D-printed resin mold was used to fabricate the microfluidic parts. The microchannel was made of polydimethylsiloxane (PDMS) material that is biocompatible. The electrodes were made of gold due to its biocompatibility and non-oxidation, and a titanium layer was sputtered as the buffer layer for the adhesion of the sputtered gold layer to the glass. A standard microfabrication process was employed to create the electrode pattern. O₂ plasma treatment yielded leakage-free bonding between the patterned glass and PDMS structure containing the microfluidic channel. The maximum voltage applied to the electrodes (26 V) was lower than the threshold value for cell electroporation. The simulations and experimental results both confirm the effectiveness of the proposed microfluidic chip.

1. Introduction

Microfluidics is a rapidly growing field that concerns the manipulation and study of minute amounts of fluids flowing through microchannels [1]. Due to its precise control over fluids, microfluidics presents unique features, including reduced sample size requirements, enhanced speed and sensitivity, and the ability to integrate multiple analytical and laboratory functions onto a single chip [2]. This interdisciplinary technology has led to astonishing advances in various domains, including drug discovery [3], medical diagnostics [4], biosensing [5], environmental monitoring [6], and food safety [7].

The ability to analyze cells is the cornerstone of numerous scientific investigations, including diagnostic and therapeutic applications and biomedical research. The study of cells provides essential information on their structure, function, and behavior, and aids in understanding the physiology of organisms. The information obtained through cell analysis has significant applications in fields like oncology, neurology, and immunology [8]. In this regard, isolating specific cell populations from a heterogeneous sample with high purity and yield is a vital step in several experimental protocols [9]. Cell separation processes are necessary for gene expression analysis, proteomics, and cell-based therapies.

The combination of microfluidics and cell separation has unlocked new prospects in studying intricate cellular processes. Based on their operational principles, microfluidic cell separation techniques can be classified into passive and active methods [10]. Passive methods harness the power of meticulous channel structures, hydrodynamic forces, and steric interactions to manipulate particles via various mechanisms, such as deterministic lateral displacement (DLD) [11], pinch flow fractionation (PFF), hydrodynamic filtration, and inertial and secondary flow [12]. Conversely, active microfluidic separation methods rely on external fields to propel particles toward specific locations for separation. Fluorescence-activated cell sorting (FACS) [13], acoustophoresis [14], magnetophoresis [15], and dielectrophoresis (DEP) [16] are some of these active methods.

One of the most prominent microfluidic cell separation techniques is the DEP separation method, a non-invasive and label-free approach. Its high flexibility ensures the selective manipulation of cells based on their diverse bio-physical characteristics, including their size, shape, and dielectric properties. Moreover, it is highly proficient in processing large volumes of cells, resulting in high-purity samples with exceptional throughput. Lastly, DEP is a highly versatile tool that can integrate with other microfluidic techniques, including microfluidic imaging, single-cell analysis, and microfluidic sensors [17,18].

In this work, we present the design and fabrication of an integrated microfluidic chip capable of separating cells of similar size based on their structural differences via the DEP mechanism. The novel feature of our microchip lies in utilizing a single applied frequency to achieve cell focusing, without using sheath flows and separation, while positioning electrodes on both sides of the microchannel. We used the negative DEP phenomenon to narrow the streamline of cells in the middle of the microchannel. Hence, all cells experienced the same electric field, with the sole differentiating factor being the structure-based variation in the DEP forces they encountered. The subtle arrangement and size of the focusing and separating microelectrodes attained optimal non-uniformity of the electric field while minimizing the voltage requirements to promote cell viability. Our easy, low-cost, and rapid fabrication method allows cost-effective mass production of our device. We simulated flow field distribution, electric field distribution, and particle trajectories to optimize the device operation. Finally, we validated our microchip’s performance by conducting experiments using live and dead yeast cells as bio-particles with similar sizes.

2. Materials and Methods

2.1. Theoretical Background

The DEP phenomenon occurs when non-uniform electric fields interact with neutral particles possessing a dipole moment. When such particles are placed in an inhomogeneous electric field, charges will start accumulating at the interface between the medium and the particle, creating dipoles that interact with the electric field. Consequently, the particles undergo a net force and begin to move. The fundamental principle of DEP relies on the difference in polarizabilities between the particles and their suspending medium. If the particle has a higher polarizability than the medium, it will experience a net force towards areas of high electric fields. Conversely, if the particle’s polarizability is lower than that of the medium, the DEP force will be directed in the opposite direction.

Utilizing the dipole moment method on a homogenous spherical particle of radius r suspended in a dielectric medium with permittivity ε_m, the time-average DEP force acting on the particle can be determined [19]:

$$〈\left.{ \overrightarrow{F}}_{DEP}(t\right)〉=2\pi {\epsilon }_m{r}^{3}Re\left\{f_{CM}\left(\omega \right)\right\}\nabla E_{rms}^2,$$

(1)

where Re { } is the real part of a complex number, E_rms is the root mean square of the applied electric field, and f_CM (ω) is the Clausius–Mossotti factor [19]:

$$f_{CM}\left(\omega \right)=\frac{{\epsilon }_p^{*}\left(\omega \right)-{\epsilon }_m^{*}\left(\omega \right) }{{\epsilon }_p^{*}\left(\omega \right)+2{\epsilon }_m^{*}\left(\omega \right)},$$

(2)

where ${{\epsilon }_p}^{*}\left(\omega \right)$and${{\epsilon }_m}^{*}\left(\omega \right)$are the effective complex permittivity of the particle and medium, respectively, which is given by$\epsilon -j\sigma /\omega$, where ε is the permittivity of the material, σ is the material conductivity, and ω is the angular frequency.

From the DEP force equation, it is evident that the DEP force direction depends on the value of Re {f_CM (ω)}. When Re {f_CM (ω)} > 0, the DEP force acts in the direction of the high electric field, causing the particle to be attracted towards the field’s region, which is called positive DEP (PDEP). Conversely, for Re {f_CM (ω)} < 0, particles are deflected from the region of higher field intensity to a region with lower field intensity, known as negative DEP (NDEP).

Using the two-shell model (Figure 1a) for live and dead yeasts [20], we plotted the Re {f_CM (ω)} (Figure 1b) and determined 10 kHz to be our optimal operating frequency.

2.2. Design and Simulation

Figure 2 presented herein illustrates our novel and inventive design strategy:

Our initial objective aimed to ensure that all cells experienced the same electric field. We leveraged the NDEP phenomenon to cause the movement of particles in the same direction toward low-field regions by applying a frequency of 10 kHz to the focusing electrodes. To facilitate the concentration process and reduce the required applied voltage, we began with a smaller section with a width of 50 microns that gradually widened to 100 microns. At the 100-micron segment, we employed two sets of four electrodes to concentrate the cells towards the center of the microchannel. The width of the upper electrodes was 120 microns (Figure 3a), while the lower electrodes had an 80-micron width, creating the desired non-uniform field. Figure 3b shows the overall performance of the focusing section consisting of three subsections, with a total length of 3400 microns.

After cell focusing, we went on to separate the cells by gradually increasing the gap between viable (green) and nonviable (red) cells using NDEP. The separation section was carefully optimized to ensure the maximum distance between the two clusters of cells, utilizing a 10:1 size ratio for the electrodes in an arrangement that caused the particles’ trajectory to oscillate about the channel’s symmetry axis. In the final part of the separation section, we incorporated a step increase in the channel width to ease separation by widening the gap between the two groups of cells. Through further simulations and optimizations, we determined that 26 volts is the minimum operating voltage for both focusing and separation electrode pairs, with the red electrodes grounded. Figure 4a displays the electric field intensity over the entire structure. Moreover, Figure 4b shows the particles’ trajectory near the outlets, with a 37-micron gap between the two cell groups.

2.3. Fabrication

Figure 5 represents the fabrication steps, comprising three phases: microelectrode formation on glass, microfluidic channel creation, and bonding. Gold was selected for the microelectrodes due to its suitable electrical conductivity, non-oxidizing properties, and biocompatibility. A sputtering process was employed to deposit a titanium layer to improve the adhesion of gold to glass, followed by Au layer deposition. To form the pattern of our microelectrodes, we performed standard photolithography by utilizing S1813 positive photoresist. Afterward, the excessive Au and Ti were etched, leaving only the microelectrode details. For the microfluidic part, we employed soft lithography, which involved using an LCD 3D printer to produce a 3D-printed mold to cast the PDMS. Subsequently, the resulting microchannel part was cured and perforated to create inlet and outlet ports. Next, we plasma-bonded the PDMS-based microchannel part to the patterned substrate containing electrodes to form an integrated microfluidic chip. Lastly, the inlet and outlet hoses were placed.

Figure 6 shows the integrated microfluidic chip.

2.4. Sample Preparation

Since we wanted to develop a yeast solution with minimal electrical conductivity, we chose deionized water as the base solvent. Initially, we heated 50 milliliters of deionized water to 40 °C for 30 min, followed by the addition of 3.5 g of sugar, which we stirred to dissolve. We then added 1.5 g of dry yeast powder and stirred the solution. The resulting solution containing cells was diluted in a 1:4 ratio with the base sugar water solution. We subjected half of the original yeast solution to a temperature of 100 °C for 30 min to obtain a solution with dead yeast cells.

Using the electrochemical impedance spectroscopy (EIS) system, we determined the relative permittivity and conductivity of the base solution to be 243.6 and 78.64 uS/cm.

3. Results and Discussion

The results presented in Figure 7a–c exhibit the successful implementation of particle focusing using the NDEP phenomenon. After entering the channel and passing through the two pairs of concentrating electrodes, each consisting of four electrodes, cells are positioned in the center of the microchannel. By exploiting the NDEP phenomenon, particles tend to migrate toward regions with lower electric field intensity, experiencing oscillatory and sinusoidal motion along the channel’s symmetry axis before ultimately settling at the microchannel centerline.

Figure 7d,e indicate the cells’ trajectory near the first and second separating electrodes, respectively. As observed, particles move away from small electrodes where electric field lines accumulate and move towards the regions of lower electric field intensity.

4. Conclusions

We introduced an integrated microfluidic chip for separating bio-particles of similar size. Our innovative microelectrode design and microchannel architecture enable the precise separation of cells based on their distinct dielectric properties. Leveraging a single frequency in our microelectrode configuration, we achieved exceptional efficiency and accuracy in sorting bio-particles. Our microfluidic chip comprises two parts—a focuser and a separator. Our NDEP force-driven focuser actively aligns particles in the center of the channel without any additional pump or sheath flow. We employed an electric field of 26 volts at a 10 kHz frequency to accomplish this task. In the separator part, we utilized the NDEP phenomenon to separate particles through varying deviations in the electric field, using the electric field conditions as the focuser part. Through a step increase in channel width, we maximized the gap between the two target particles to improve the effectiveness of the separation process. Our microdevice is easy to fabricate, requires low voltage, and is compatible with many types of cells, making it an attractive option for research and clinical applications. Our novel chip design has the potential to drastically change existing cell separation methods, opening the door for further advancements in various fields of life sciences and biomedical research.