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Integrated Dual Nanoprobe-Microfluidic System for Single Cell Penetration

Abdul Hafiz Mat Sulaiman1 and Mohd Ridzuan Ahmad1,2 , 1Dept. of Control and Mechatronic Engineering, Faculty of Electrical Engineering,

Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. 2Institute of Ibnu Sina, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

[email protected] and [email protected]

Abstract—This paper presents the results in single cell intracellular penetration using the integrated dual nanoprobe-microfluidic system. Current technique for single cell penetration is using a manual controlled nanomanipulator which been attached with a nanoprobe. Due to this, successful single cell penetration depends on the operator skills. We proposed an integrated dual nanoprobe-microfluidic system. The system was being developed to reduce the requirement for skilled operator in single cell electrical measurement. We tested the system function for single cell penetration via finite element simulation. Based on the results, the single cell can be penetrated at fluid velocity of 5.3 µm/s. Currently, the developed system is suitable for cell viability detection application. However, this system has the potential to be used in single cell thermal measurement, single cell drug delivery, and early disease diagnosis.

Index Terms—System integration, nanoprobe, microfluidic, electrical measurement

I. INTRODUCTION Single cell analysis has gained researcher attention in

microbiological studies thanks to the rapid development of nanotechnology. The cells are now being studied individually and not only based on populations of cells. The main advantage of single cell analysis over population analysis is accuracy.

Population studies unable to characterize individual cell accurately as the result obtain based only on average data. Each cell may have unique properties which could be used as a marker for cell type classification. Each cell type is expected to be differentiated from one another if their individual properties, i.e. mechanical, electrical, and chemical, can be characterized. This information is important in early disease detection applications. Beside mechanical properties [1-3], cells can be characterized based on the electrical properties .

In recent years, studies on single cell analysis have been focusing on characterizing the cells dielectric properties, i.e. dielectric constant and conductivity [4-9]. Some of the research have shown its potential in a practical application, i.e. single cell viability detection [10] and single cell cancer detection [11].

Even though the single cell electrical measurement devices have already been invented, most of the devices have several disadvantages, i.e. labour intensive and bulky system. These

disadvantages make the single cell electrical measurement inaccurate, slow throughput rate, and high operation cost.

Operator skills play an important role where each measurement is done manually and new operator need to be intensively trained for an accurate measurement. While, bulky system makes the single cell measurement less portable and can only perform in a restricted area, e.g. clean room.

Therefore, there is a need to reduce the operator role in single cell measurement for a consistent measurement accuracy and higher throughput rate and also improve the device from bulky system to a more portable system. In the end, single cell electrical measurement can be conducted not only by inexperienced operator but also under none specific lab environment. We proposed an integrated dual nanoprobe-microfluidic system (IDNMS) for reducing the requirement for labour skills, higher throughput rate and portable system.

This paper presents the single cell penetration results using the developed IDNMS. Section II in this paper explains the integrated dual nanoprobe-microfluidic system concept and features. Section III discussed on the methodology used in testing IDNMS for single cell passive penetration capability. Lastly, section IV discusses the results obtained from the simulation.

II. INTEGRATED DUAL NANOPROBE-MICROFLUIDIC SYSTEM

The idea for dual nanoprobe-microfluidic system integration was inspired by the fast growing use of microfluidic in assisting single cell studies. Previous experimental work on using dual nanoprobe for single cell intracellular penetration used an active approach where the dual nanoprobe was being navigated by a nanomanipulator to penetrate the single cell which in a static position on a substrate [10].

In contrary, the new integrated dual nanoprobe-microfluidic system is using a passive approach where the cell is being forced to be penetrated by a fixed dual nanoprobe in a microchannel. Figure 1 shows the concept for the passive approach. Basically, the target single cell will be forced to move due to fluid flow in the narrow channel and penetrated by the dual nanoprobe up to the intracellular part of the cell. A similar concept has been used in single cell microinjection for drug delivery [12]. Even though this technique is invasive, the

2013 IEEE International Conference on Control System, Computing and Engineering, 29 Nov. - 1 Dec. 2013, Penang, Malaysia

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cell is expected to recover from its small wound due to the small size of the probe [13, 14].

The integrated dual nanoprobe-microfluidic chip design has several features. Figure 2 shows the overview for integrated dual nanoprobe-microfluidic system. The chip has a pair of Tungsten electrode which connecting the dual nanoprobe with the measurement equipment. Dual nanoprobe is positioned in the sensor area. There are two channel outlets (suction and cell flush) and one cell inlet. The inlet and outlet guide the cell into and out of the chip using a micro pump. The micro pump performs infusion and withdraws on the fluid inside the chip.

Figure 3 shows the complete measurement setup using IDNMS for single cell electrical property characterization. The integrated dual nanoprobe-microfluidic chip requires other equipment before single cell electrical property characterization can be conducted. There are three steps of the procedure need to be performed, i.e. fluid manipulation, single cell observation, and impedance measurement. The fluid flow rate inside the chip is control by the external pump. The pump type can be changed depending on the flow rate requirement.

Single cell inside the microfluidic chip can be observed using an inverted microscope. This type of microscope is suitable for observation for a single cell. The chip itself has been designed as a transparent chip to accommodate with the inverted microscope viewing requirement.

Electrical measurement requires an Electrochemical Impedance Spectroscopy (EIS) system that is connected to the

electrode on the chip. EIS will measure the impedance of the single cell. All the equipment can be integrated into one complete measurement system when connected to a central computer which enables the measurement to be conducted by only one person.

III. METHODOLOGY The developed integrated dual nanoprobe-microfluidic

system has being tested via finite element analysis software. The main objective of this study is to determine the IDNMS capability for passive single cell penetration.

Basically, this study has been divided into two stages. The first stage is on single cell modeling. The single cell has been modeled based on Saccharomyces cerevisiae or yeast cell and was validated through experimental data comparison. The second stage focus on flow rate optimization for single cell penetration using the modeled single cell.

A. Single Cell Mechanical Model In this research, the single cell mechanical model was

created based on experimental data obtained in single cell mechanic characterization using nanoneedle [15]. Figure 4 shows the mechanical model for a single cell. The single cell

Cell

A

Microfluidic channel 4 µm

Ammeter DC Voltage

Dual nanoprobe

Flow direction

Fig. 1. Concept overview

40 mm

Fig. 2. Integrated dual nanoprobe-microfluidic system overview.

Fig. 3. Measurement Setup

Micro Pump

Inverted Microscope

Electrochemical Impedance

Spectroscopy (EIS)

Computer

Microfluidic Chip

Fig. 4. Single cell mechanical model

4 µm Cytoplasm

(Homogenous fluid)

Cell wall 207 µm


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was modelled as a 3D solid deformable C3D8R and 8-node linear brick 3D stress element of a two layer sphere, i.e. cell wall layer and cytoplasm layer, with the size of 4 µm in diameter. The actual yeast cell size varies from 4-6 µm [16]. The cell wall material was defined using data extracted from the nanoneedle experiment with thickness of 207 nm [17, 18] and the cytoplasm was defined as a homogenous fluid with a density of 1084 kg/m3 [19].

In order to validate the created cell model, we simulate the experimental nanoneedle technique for single cell stiffness measurement. Then, we compared single cell penetration force and deformation between simulation result and experiment data. This is one of the approaches for simulation model validation [20]. Figure 5 shows the nanoneedle technique for measuring single cell stiffness simulation setup. The simulated technique consists of a cantilever, nanoneedle, and single cell model.

Basically, the cantilever that attached with nanoneedle will move down to perform indentation on the cell single cell which has been placed on a substrate. Nanoneedle will eventually penetrate the cell wall at one point. Cantilever deflection is being measured to calculate for the indentation force.

B. Single Cell Passive Penetration Fluid flow rate inside the microfluidic chip play a major

role in single cell penetration. It is essential to apply the correct flow rate in order to avoid excessive single cell penetration. Fluid flow rate in a microfluidic, Q can be calculate using equation 1 given as (1) where A is the cross section area of a channel and v is the fluid velocity. The unit for flow rate is litre per minute (l/min). Basically, flow rate is the total volume of fluid that passes through a cross section in one minute.

The integrated dual nanoprobe-microfluidic chip has a fixed channel cross section which was being set to allow only a

single cell to pass through. Therefore, only fluid velocity is being manipulated in the optimization process. The velocity was being optimized through simulation. The velocity is considered sufficient if the dual nanoprobe is able to penetrate the single cell without excessive damage to the cell.

Figure 6 shows the simulation setup for single cell passive penetration. The simulation consists of the single cell mechanical model, dual nanoprobe and microchannel. Figure 5 shows the simulation setup. In this simulation the flow rate is being controlled by applying initial velocity for both cell and fluid. Once started, the velocity will remains constant until the cell gets in contact with the dual nanoprobe. The cell is being penetrated depends on the applied velocity. The velocity is considered sufficient if the dual nanoprobe is able to penetrate the single cell without excessive damage to the cell.

IV. RESULTS AND DISCUSSION

A. Single Cell Mechanical Model Validation Results There are two criteria for single cell mechanical model

evaluation, i.e. penetration force and cell deformation. These criteria are essential in predicting integrated dual nanoprobe-microfluidic system performance in the actual fabricated system. Figure 7 shows the results for single cell indentation using nanoneedle. At a glance, the simulation shows a good visual result in penetrating the cell. The cantilever deflects when the nanoneedle became in contact with the cell and the cell seems to resist the nanoneedle indentation. Eventually, the nanoneedle succeeds in penetrating the cell wall at one point

Fig. 6. Single cell penetration setup.

Nano probe

Single cell

Apply Velocity

Microchannel (Filled with fluid element)

Nano probe 4 µm

Fig. 5. Single cell mechanical model validation

Cantilever

Nanoneedle

Single cell

Fig. 7. Simulation results of single cell mechanical model validation (a) t = 0 s (b) t = 6 s (c) t = 6.1 s

(a) (b)

(c)


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and reached the cell internal part or cytoplasm. Even though result observation is one of the techniques for validation, it requires a group of expert examiners which to help determine a ‘realistic’ cell model.

Quantitative values for penetration force and cell deformation were extracted from the experimental and plotted on a graph. Figure 8 shows the force-cell deformation for both simulation and experiment. The penetration force of 640 nN lies within the reported experimental range of 550-778 nN [15]. However, the cell deformation in the simulation is higher than the experimental data. Cell deformation can vary depends on many factors, e.g. indentation speed and environment. However, the difference for the cell deformation between simulation and experiment is due to the cell shape. The cell model may not able to resemble the actual cell shape on a substrate due to limited information. However, the mechanical cell model satisfied the requirement for this research by showing similar penetration force.

B. Single Cell Passive Penetration Results Figure 9 shows the single cell penetration simulation

results inside IDNMS. Based on the results obtained, the integrated dual nanoprobe-microfluidic system able to penetrate single cell. At velocity of 5.3 µm/s, the cell is able to be penetrated by the dual nanoprobe. This value was obtained through flow rate optimization where the initial velocity was set at 5.0 µm/s. However, at this velocity the cell is impenetrable and only deform. Then, the velocity was being increased to 5.3 µm/s. At this velocity, the dual nanoprobe was able to penetrate the single cell successfully without excessive damage to the cell. The velocity was being increased further. At a velocity of 5.5 µm/s the dual nanoprobe was starting to penetrate deeper than it should and at velocity 6.0 µm/s the cell was excessively been penetrated. Table I summarize the simulation results. The suitable velocity range is in between 5.3-5.5 µm/s.

The cell damage can be reduced by applying a suitable flow rate and increase the cell chances for recovery. This is important for conducting multiple measurements on the same cell.

V. CONCLUSION We performed a simulation on single cell penetration using

integrated dual nanoprobe-microfluidic system for single cell electrical property measurement. The single cell was modeled and validated before it is been used to test the integrated dual nanoprobe-microfluidic system capability. From the results, the integrated dual nanoprobe-microfluidic system able to penetrate the single cell by controlling the fluid flow rate inside a microchannel. The optimized velocity for the cell was found to be in the range of 5.3-5.5 µm/s.

ACKNOWLEDGMENT We would like to express our appreciation towards the

Ministry of Higher Education Malaysia (MOHE) grant no. 78677 (FRGS), (MOHE) grant no. 4L038 (ERGS) and Universiti Teknologi Malaysia, grant nos. 77973 (NAS), 03H80 (GUP) and 02H34 (GUP) for funding this project and Micro Nano Mechatronic research group members for their endless support.

Fig. 9. Simulation results of single cell passive penetration at fluid velocity of 5.3 µm/s. (a) t = 0 s (b) t = 0.25 s (c) t = 0.45 s (d) t = 1 s

(a) (b)

(d) (c)

Fig. 8. Cell deformation-force results comparison.

Penetration force region

TABLE I. FLOW RATE OPTIMIZATION

No. Fluid velocity, µm/s

Flow rate, pl/min

Penetration Status

1 5.0 4.8 No

2 5.3 5.1 Yes

3 5.5 5.3 Yes

4 6.0 5.8 Yes (Excessively)


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