*1.2.4 Impedance measurement procedure*

Standard short and open self-calibration procedure has been used for impedance analyzer in order to perform the impedance measurement. Furthermore, to calibrate the chip, impedance of 1xPBS solution was measured at the 20 μm of electrode gap. Three microfluidic devices were utilized for reproducibility testing and the experiments were conducted at room temperature. To validate the equivalent circuit model, impedance of the medium between microneedle was measured. The solutions were sterilized DI water and PBS (Photosphate-buffered saline) with conductivities 6 and 1.4 S/m respectively.

Initially, 1 ml of PBS with concentrations of 1500 mOsm was prepared for the chip cleaning process. The sample was loaded into a syringe and driven through the microchannel using a syringe pump with the flow rate of syringe pump was kept constant (60 μl/min). After flushing with PBS solution, yeast cell of 1 ml of each seven different concentrations of sample from 102 to 10<sup>9</sup> cfu/ml were driven

**Figure 2.** *The experimental set-up diagram.*

through the microchannel at a flow rate of 6 μl/min. The impedance of each solution was measure by connecting the microneedles with impedance analyzer. Then, an AC signal frequency range from 100 Hz to 5 MHz with an applied voltage of 1 V was apply to determine the impedance spectra (impedance and phase vs. frequency) in order to differentiate the variations of solution samples. Between each sample measurement, the microchannel chip was flushed by DI and PBS water for 1 and 2 minutes respectively. The impedance analyzer (Hioki IM3570) GUI and post-processed in MATLAB (MathWorksInc, USA) was utilized to record the data. The impedance change during the passage yeast cells at sensing area was measured. In order to monitor the behavior of impedance for each sample, the impedance value at three frequencies (100 kHz, 500 kHz and 1 MHz) was measured.

Single cell detection and measurement was conducted based on impedance measurement with or without single cell at the sensing area. Two sample of microbeads with diameter 15 and 9 μm suspended in 1 ml of PBS with concentration of 103 per ml were utilized to perform this measurement and detection. Each sample were driven through the microchannel at a flow rate of 6 μl/min and measured using an AC signal frequency range from 100 Hz to 5 MHz.

#### **1.3 Result and discussion**

As a proof of concepts, the dependencies of the impedance on the various concentrations of yeast cells and a single microbead in the suspension medium by using this microfluidic device are studied. **Figure 3** presents the measured impedance spectra and fitting spectra (on a log scale) of the system for two of microchannel filled with sterilized DI water and PBS at frequency range 1 kHz to 1 MHz. For simulation, 100 data points on the impedance measured spectrum were used as input to the equivalent circuit [see **Figure 1(b)**] and generating the fitting impedance spectrum by using MATLAB. For high conductivity fluid (PBS), the result shows two domains which were an electrical double layer (EDL) region and a resistive region [24]. The EDL occurred in the low frequency range from 1 kHz to approximately 300 kHz, whereas the resistive region occurred in high frequency from 300 kHz to 1 MHz. The agreement between the measured and fitting spectra result indicated that our developed circuit model for this system is feasible to determine the impedance characteristics of solution medium.

**107**

**Figure 4.**

*at 1 MHz with linear relationship.*

*Microfluidic Device for Single Cell Impedance Characterization*

water with the different cell concentration in the range 104

DI water as a reference. After washing the microchannel, 108

*(a) Impedance spectra of yeast cells in water with cell concentrations ranging from 102*

*DI water as controls; (b) the logarithmic value of the concentration of yeast cells and the impedance measured* 

To illustrate the cell detection capability of the device, yeast cell and microbeads with different concentration was utilized. Yeast cells concentration ranging from

impedance by referring the impedance of DI water as a control. Afterward, the microchannel chip was washed by the PBS followed by DI water at maximum flow

The maximum flow rate of the liquid can flow inside microchannel without leaking is 300 μl/min. **Figure 4(a)** shows the impedance spectra of yeast cell in DI

the microchannel resulting in an increase in impedance. It can be seen the impedance spectra of yeast cell in DI water across the sensing area (two microneedles)

 cfu/ml were infused inside microchannel with fixed flow rate 6 μl/min and fixed electrode gap (25 μm). A sweep frequency (100 kHz to 5 MHz) AC signal (1 V) was applied to the one side of the microneedle and the current entering at another side of microneedle was measured to calculate the impedance of concentra-

cfu/ml was injected resulting in a drop-in

–109

cfu/ml, along with

cfu/ml was infused to

 *to 109*

*cfu/ml, along with* 

*DOI: http://dx.doi.org/10.5772/intechopen.90657*

tion of yeast cells in DI water. Initially, 109

102 to 109

rate.

**Figure 3.** *Impedance spectra of sample solution together with their fitting spectra: (a) DI water (b) PBS.*

#### *Microfluidic Device for Single Cell Impedance Characterization DOI: http://dx.doi.org/10.5772/intechopen.90657*

*Current and Future Aspects of Nanomedicine*

was measured.

**1.3 Result and discussion**

through the microchannel at a flow rate of 6 μl/min. The impedance of each solution was measure by connecting the microneedles with impedance analyzer. Then, an AC signal frequency range from 100 Hz to 5 MHz with an applied voltage of 1 V was apply to determine the impedance spectra (impedance and phase vs. frequency) in order to differentiate the variations of solution samples. Between each sample measurement, the microchannel chip was flushed by DI and PBS water for 1 and 2 minutes respectively. The impedance analyzer (Hioki IM3570) GUI and post-processed in MATLAB (MathWorksInc, USA) was utilized to record the data. The impedance change during the passage yeast cells at sensing area was measured. In order to monitor the behavior of impedance for each sample, the impedance value at three frequencies (100 kHz, 500 kHz and 1 MHz)

Single cell detection and measurement was conducted based on impedance measurement with or without single cell at the sensing area. Two sample of microbeads with diameter 15 and 9 μm suspended in 1 ml of PBS with concentration of 103

ml were utilized to perform this measurement and detection. Each sample were driven through the microchannel at a flow rate of 6 μl/min and measured using an

As a proof of concepts, the dependencies of the impedance on the various concentrations of yeast cells and a single microbead in the suspension medium by using this microfluidic device are studied. **Figure 3** presents the measured impedance spectra and fitting spectra (on a log scale) of the system for two of microchannel filled with sterilized DI water and PBS at frequency range 1 kHz to 1 MHz. For simulation, 100 data points on the impedance measured spectrum were used as input to the equivalent circuit [see **Figure 1(b)**] and generating the fitting impedance spectrum by using MATLAB. For high conductivity fluid (PBS), the result shows two domains which were an electrical double layer (EDL) region and a resistive region [24]. The EDL occurred in the low frequency range from 1 kHz to approximately 300 kHz, whereas the resistive region occurred in high frequency from 300 kHz to 1 MHz. The agreement between the measured and fitting spectra result indicated that our developed circuit model for this system is feasible to

AC signal frequency range from 100 Hz to 5 MHz.

determine the impedance characteristics of solution medium.

*Impedance spectra of sample solution together with their fitting spectra: (a) DI water (b) PBS.*

per

**106**

**Figure 3.**

To illustrate the cell detection capability of the device, yeast cell and microbeads with different concentration was utilized. Yeast cells concentration ranging from 102 to 109 cfu/ml were infused inside microchannel with fixed flow rate 6 μl/min and fixed electrode gap (25 μm). A sweep frequency (100 kHz to 5 MHz) AC signal (1 V) was applied to the one side of the microneedle and the current entering at another side of microneedle was measured to calculate the impedance of concentration of yeast cells in DI water. Initially, 109 cfu/ml was injected resulting in a drop-in impedance by referring the impedance of DI water as a control. Afterward, the microchannel chip was washed by the PBS followed by DI water at maximum flow rate.

The maximum flow rate of the liquid can flow inside microchannel without leaking is 300 μl/min. **Figure 4(a)** shows the impedance spectra of yeast cell in DI water with the different cell concentration in the range 104 –109 cfu/ml, along with DI water as a reference. After washing the microchannel, 108 cfu/ml was infused to the microchannel resulting in an increase in impedance. It can be seen the impedance spectra of yeast cell in DI water across the sensing area (two microneedles)

#### **Figure 4.**

*(a) Impedance spectra of yeast cells in water with cell concentrations ranging from 102 to 109 cfu/ml, along with DI water as controls; (b) the logarithmic value of the concentration of yeast cells and the impedance measured at 1 MHz with linear relationship.*

increase with decreasing the cell concentration of cells [14]. According to the observation result, it can be said that cell suspension with high concentrations is more conductive than those with lower concentrations.

The conductivity of solution varies proportionally to the number of cell concentration at fixed volume of solution [25]. In some studies, the relative dielectric permittivity and charged polyelectrolytes inside the cell also may affect the impedance of solution [14]. The optimum region for sensing microneedle to differentiate the cell concentration in DI water occurs between 500 kHz to 5 MHz. The impedance values of the suspensions in this frequency region were significantly different from each other. The experiment was repeated two times measurement cycle and showed the similar result.

In cell detection experiment, frequency lower than 100 kHz are not considered, as the EDL started to influence the measurement at frequency below 300 kHz [17, 26]. In order to investigate the relationship between impedance value and cell concentration, we selected 1 MHz as the best representative frequency. **Figure 4(b)** illustrates the impedance responses of the sample containing different yeast cell concentrations and DI water at frequency measurement 1 MHz. The impedance of the solution was significantly increased from 207.63 to 225.42, 247.61, 284.48, 314.64, and 348.51 kΩ when the yeast concentration decreasing from 109 to 108 , 107 , 10, 10 and 104 cfu/ml respectively. After the cell concentrations were lower than 104 cfu/ml, impedance value shows no significant changing between each other or DI water. In additions, the pattern of the result shows a linear relationship between the impedance and the logarithmic value of the cell concentration at cell concentration from 104 to 109 cfu/ml (see **Figure 4**). The linear regression equation of this result is Z (kΩ) = 58.3 log X (cells/ml) + 175.4 with R2 = 0.986. The detection limit was calculated to be 1.2 × 104 cfu/ml. Error bars are standard deviations of five measurements cycle.

In order to measure the cell concentration in DI water suspensions, the linear regression equation of the impedance of the yeast suspensions was used. This device can be utilized to quantify cells in suspensions other than impedance microbiology and impedance biosensors for bacteria detection since the detection limit of this method is comparable with another sensor. The reported sensor for detection of pathogenic bacteria are QCM immunosensors for detection of Salmonella with detection limits of 9.9 × 105 cfu/ml [27], surface plasmon resonance (SPR) sensor for detection of *E. coli* O157:H7 with a detection limit of 107 cfu/ml [28] and SPR immounosensors for detection of Salmonella enteritidis and Listeria monocytogens with detection limits of 106 cfu/ml [29].

In order to demonstrate the capability of this device in detecting the present of single cell, two size of micro bead have been flowing inside the microfluidic device. The impedance of PBS solution as a control was initially infused inside the microfluidic device. Then two samples of microbeads in PBS solution were infused inside the microchannel with the same flow rate and electrode gap of yeast cell concentration measurement. **Figure 5(a)** shows the 15-μm microbead flow through the sensing area and a sweep frequency ranging from 100 kHz to 3 MHz AC signal (1 V) was applied to the electrode. As the result, the impedance spectrum is plotted over the field frequency, as shown in **Figure 5(b)**. The figure shows the average electrical impedance data for two size of beads and PBS solution without present of beads. From this average data, it is expected that the electrical impedance spectrum can be used to differentiate between the sizes of beads. The beads (9 and 15 μm in diameter) are clearly discriminated by impedance spectrum. The impedance increases with the increasing of the size of particle. Due to the presence of a single bead that can be regarded as an insulating object, the electrical resistance of the sensing channel was slightly increased.

**109**

**Figure 5.**

*Microfluidic Device for Single Cell Impedance Characterization*

As the result, we conclude this device can detect the cell concentrations in solution medium and the single microbead at the high frequency range between 100 kHz and 5 MHz. In this experiment, we did not determine the detection capability at the frequency lower than 100 kHz. For the future work, we will focus on the measurement and detection to the human cell (normal and cancer cell) the size of microneedle, single cell detection and utilize a non-polarizable electrode, that is, Ag/AgCl (to eliminate the EDL), in order to improve the performance of the device.

*(a)The single microbead with diameter of 15 μm flows through the sensing area. (b) Impedance spectrum of* 

*two different sizes of beads in PBS solution and PBS solution (without bead).*

*DOI: http://dx.doi.org/10.5772/intechopen.90657*

#### *Microfluidic Device for Single Cell Impedance Characterization DOI: http://dx.doi.org/10.5772/intechopen.90657*

*Current and Future Aspects of Nanomedicine*

showed the similar result.

10, 10 and 104

tion from 104

to 109

was calculated to be 1.2 × 104

detection limits of 9.9 × 105

with detection limits of 106

sensing channel was slightly increased.

measurements cycle.

104

more conductive than those with lower concentrations.

increase with decreasing the cell concentration of cells [14]. According to the observation result, it can be said that cell suspension with high concentrations is

The conductivity of solution varies proportionally to the number of cell concentration at fixed volume of solution [25]. In some studies, the relative dielectric permittivity and charged polyelectrolytes inside the cell also may affect the impedance of solution [14]. The optimum region for sensing microneedle to differentiate the cell concentration in DI water occurs between 500 kHz to 5 MHz. The impedance values of the suspensions in this frequency region were significantly different from each other. The experiment was repeated two times measurement cycle and

In cell detection experiment, frequency lower than 100 kHz are not considered,

cfu/ml respectively. After the cell concentrations were lower than

cfu/ml (see **Figure 4**). The linear regression equation of this

cfu/ml. Error bars are standard deviations of five

cfu/ml [27], surface plasmon resonance (SPR) sensor

 cfu/ml, impedance value shows no significant changing between each other or DI water. In additions, the pattern of the result shows a linear relationship between the impedance and the logarithmic value of the cell concentration at cell concentra-

result is Z (kΩ) = 58.3 log X (cells/ml) + 175.4 with R2 = 0.986. The detection limit

In order to measure the cell concentration in DI water suspensions, the linear regression equation of the impedance of the yeast suspensions was used. This device can be utilized to quantify cells in suspensions other than impedance microbiology and impedance biosensors for bacteria detection since the detection limit of this method is comparable with another sensor. The reported sensor for detection of pathogenic bacteria are QCM immunosensors for detection of Salmonella with

immounosensors for detection of Salmonella enteritidis and Listeria monocytogens

In order to demonstrate the capability of this device in detecting the present of single cell, two size of micro bead have been flowing inside the microfluidic device. The impedance of PBS solution as a control was initially infused inside the microfluidic device. Then two samples of microbeads in PBS solution were infused inside the microchannel with the same flow rate and electrode gap of yeast cell concentration measurement. **Figure 5(a)** shows the 15-μm microbead flow through the sensing area and a sweep frequency ranging from 100 kHz to 3 MHz AC signal (1 V) was applied to the electrode. As the result, the impedance spectrum is plotted over the field frequency, as shown in **Figure 5(b)**. The figure shows the average electrical impedance data for two size of beads and PBS solution without present of beads. From this average data, it is expected that the electrical impedance spectrum can be used to differentiate between the sizes of beads. The beads (9 and 15 μm in diameter) are clearly discriminated by impedance spectrum. The impedance increases with the increasing of the size of particle. Due to the presence of a single bead that can be regarded as an insulating object, the electrical resistance of the

for detection of *E. coli* O157:H7 with a detection limit of 107

cfu/ml [29].

to 108

cfu/ml [28] and SPR

, 107 ,

as the EDL started to influence the measurement at frequency below 300 kHz [17, 26]. In order to investigate the relationship between impedance value and cell concentration, we selected 1 MHz as the best representative frequency. **Figure 4(b)** illustrates the impedance responses of the sample containing different yeast cell concentrations and DI water at frequency measurement 1 MHz. The impedance of the solution was significantly increased from 207.63 to 225.42, 247.61, 284.48, 314.64, and 348.51 kΩ when the yeast concentration decreasing from 109

**108**

As the result, we conclude this device can detect the cell concentrations in solution medium and the single microbead at the high frequency range between 100 kHz and 5 MHz. In this experiment, we did not determine the detection capability at the frequency lower than 100 kHz. For the future work, we will focus on the measurement and detection to the human cell (normal and cancer cell) the size of microneedle, single cell detection and utilize a non-polarizable electrode, that is, Ag/AgCl (to eliminate the EDL), in order to improve the performance of the device.

#### **Figure 5.**

*(a)The single microbead with diameter of 15 μm flows through the sensing area. (b) Impedance spectrum of two different sizes of beads in PBS solution and PBS solution (without bead).*
