**1. Introduction**

The single cell analysis (SCA) has been emphasized to provide biologists and scientists to peer into the molecular machinery of individual cells. For the application of medical diagnosis, detection of cancer cells and pathogenic bacteria cells in blood is utilized as a diagnosing infectious disease. It is reported that detection of circulating tumor cells (CTCs) in the blood has shown to be clinically important for early stage metastasis or recurrence of cancer. The presence of rare CTCs in blood is ranging from only 1–100 CTCs/ml blood [1]. *Plasmodium falciparum* malaria, which kills mainly children in developing countries infected the blood sample of patients at concentration of ~1/50 μl of blood [2]. Nowadays, the analysis of single cell in biological measurements and medical research has emerged as a distinct new field and acknowledged to be one of the fundamental building blocks of life [3]. Amongst of various single cell analysis, cell impedance measurement has become an effective method of biological measurement [4]. The physiological behavior of the cells and their corresponding molecular expressions have significant effect on the cell membrane and cytoplasm conductivity and dielectric constant, which in turn affects the overall impedance characteristics [5]. For that reason, the impedance

measurements on single cells can provide relevant information about its functional status and may be a simple and significantly less complex alternative to detailed molecular expression studies.

The classical method for cell detection in suspension is using flow cytometry, which is rapid and highly accurate measurement technique. Impedance flow cytometry is an indirect signal extraction from the single cells on microchannel sensing area without having direct access into intracellular region of the cells [6]. These techniques were first reported by Coulter [7] has emerged in the microfabrication device in order to analyze microscale particle with high sensitivity. However, flow cytometry involves expensive manufacturing and labelling of the cells with fluorescent antibodies [8]. Recently, the impedance flow cytometry (IFC) has gained attention for the significant promising techniques to replace and overcome the limitations associated with flow cytometry. The IFC is preferable because of fast, real-time, and non-invasive methods for biological detection. This technique is capable to be utilized as cell counting [8], cancer cell detection [9] and bacteria detection [10]. Some groups have demonstrated detection and counting of cells by using a microfluidic integrated with electrode for various electrical measurement methods in an application of food safety [11] and real-time monitoring bio-threat [12]. This measurement technique is based on the alteration of impedance across a measurement electrode due to the blocking of ionic current passing between electrodes when a presence of the cells.

The IFC is capable to distinguish and count lymphocytes, monocytes and neutrophils in human whole blood [8]. Other studies reported that IFC can detect the presence of cells based on probing the impedance inside the cell at frequency greater than 1 MHz [13]. Fabricated nanoneedles probe inside microfluidic was utilized for measuring the presence of cells at sensor surface and making it sensitive to the dielectric properties of solution [14]. However, this device requires patterning of electrode or probe on the substrate resulting in higher cost of the fabrication process. Another limitation also needs to consider is time-consuming cleaning process of the device. Several groups have demonstrated the technique to reduce the cost of microfabrication of electrodes by using printed circuit board (PCB) as a measurement electrode. They demonstrated contactless conductivity detection in capillary electrophoresis manners [15] and cell manipulation using dielectophoresis [16]. Recently, the contactless impedance cytometry was developed to reduce the fabrication cost of impedance cytometry device [17, 18]. The electrode was fabricated on the PCB substrate (reusable component) and the thin bare dielectric substrate bonded to a PDMS microchannel (disposable component) was placed onto PCB substrate. The sensitivity of this device is the limitation since the electric field was buried in dielectric substrate and not reaches the electrolyte. Several designs and method in IFC in order to detect and analyze a cell have been reported [19, 20].

This chapter discusses a novel integrated microneedles-microfluidic system for detecting yeast cell concentration in suspension as well as detecting a single particle based on the impedance measurement. The development of the device focuses on reducing the fabrication cost while preserving the main functionality, that is, cell detection. The significant fabrication cost reduction in this work is by replacing the microfabrication of electrodes by the microneedles. This device utilized a Tungsten microneedle as a measurement electrode which can be reused and easily to be cleaned. The two microneedles were placed at half height disposable microchannel to detect and enable impedance measurement of passing cells through the applied electric field. **Figure 1(a)** illustrated the schematic diagram of the proposed microfluidic chip which consists of two microneedles integrated at both sides of the microchannel. The main sensing area microchannel length, width and thickness are

**103**

**1.1 Principle**

*equivalent circuit model.*

**Figure 1.**

*Microfluidic Device for Single Cell Impedance Characterization*

100, 25 and 25 μm respectively. The device is suitable for early cancer cell detection application in developing countries since it significantly reduces the fabrication

*(a) 3D schematic diagram of the microfluidic device structure integrated with microneedle and top view of sensing area which the impedance measurement of single particle be measured. (b) Microfluidic sensing area* 

Ohm's Law has been use as the basic principle of detecting suspended biological cells in the media. The passing cells across the sensing area have been measure by an AC current with a frequency sweep to determine the changing impedance value of media. **Figure 1(b)** illustrated the equivalent circuit model for obtain the all parameter involved in order to characterize the electrical properties of cell between two microneedles in the suspension media. The sensing area of mirofluidic chip can be modeled electrically as cell impedance of resistance Rp and cell capacitance Cp in parallel with the impedance contributed by all materials between the two

cost, that is, not required the fabrication of micro electrode.

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

#### **Figure 1.**

*Current and Future Aspects of Nanomedicine*

electrodes when a presence of the cells.

molecular expression studies.

measurements on single cells can provide relevant information about its functional status and may be a simple and significantly less complex alternative to detailed

The classical method for cell detection in suspension is using flow cytometry,

which is rapid and highly accurate measurement technique. Impedance flow cytometry is an indirect signal extraction from the single cells on microchannel sensing area without having direct access into intracellular region of the cells [6]. These techniques were first reported by Coulter [7] has emerged in the microfabrication device in order to analyze microscale particle with high sensitivity. However, flow cytometry involves expensive manufacturing and labelling of the cells with fluorescent antibodies [8]. Recently, the impedance flow cytometry (IFC) has gained attention for the significant promising techniques to replace and overcome the limitations associated with flow cytometry. The IFC is preferable because of fast, real-time, and non-invasive methods for biological detection. This technique is capable to be utilized as cell counting [8], cancer cell detection [9] and bacteria detection [10]. Some groups have demonstrated detection and counting of cells by using a microfluidic integrated with electrode for various electrical measurement methods in an application of food safety [11] and real-time monitoring bio-threat [12]. This measurement technique is based on the alteration of impedance across a measurement electrode due to the blocking of ionic current passing between

The IFC is capable to distinguish and count lymphocytes, monocytes and neutrophils in human whole blood [8]. Other studies reported that IFC can detect the presence of cells based on probing the impedance inside the cell at frequency greater than 1 MHz [13]. Fabricated nanoneedles probe inside microfluidic was utilized for measuring the presence of cells at sensor surface and making it sensitive to the dielectric properties of solution [14]. However, this device requires patterning of electrode or probe on the substrate resulting in higher cost of the fabrication process. Another limitation also needs to consider is time-consuming cleaning process of the device. Several groups have demonstrated the technique to reduce the cost of microfabrication of electrodes by using printed circuit board (PCB) as a measurement electrode. They demonstrated contactless conductivity detection in capillary electrophoresis manners [15] and cell manipulation using dielectophoresis [16]. Recently, the contactless impedance cytometry was developed to reduce the fabrication cost of impedance cytometry device [17, 18]. The electrode was fabricated on the PCB substrate (reusable component) and the thin bare dielectric substrate bonded to a PDMS microchannel (disposable component) was placed onto PCB substrate. The sensitivity of this device is the limitation since the electric field was buried in dielectric substrate and not reaches the electrolyte. Several designs and method in IFC in order to detect and analyze a cell have been

This chapter discusses a novel integrated microneedles-microfluidic system for detecting yeast cell concentration in suspension as well as detecting a single particle based on the impedance measurement. The development of the device focuses on reducing the fabrication cost while preserving the main functionality, that is, cell detection. The significant fabrication cost reduction in this work is by replacing the microfabrication of electrodes by the microneedles. This device utilized a Tungsten microneedle as a measurement electrode which can be reused and easily to be cleaned. The two microneedles were placed at half height disposable microchannel to detect and enable impedance measurement of passing cells through the applied electric field. **Figure 1(a)** illustrated the schematic diagram of the proposed microfluidic chip which consists of two microneedles integrated at both sides of the microchannel. The main sensing area microchannel length, width and thickness are

**102**

reported [19, 20].

100, 25 and 25 μm respectively. The device is suitable for early cancer cell detection application in developing countries since it significantly reduces the fabrication cost, that is, not required the fabrication of micro electrode.

### **1.1 Principle**

Ohm's Law has been use as the basic principle of detecting suspended biological cells in the media. The passing cells across the sensing area have been measure by an AC current with a frequency sweep to determine the changing impedance value of media. **Figure 1(b)** illustrated the equivalent circuit model for obtain the all parameter involved in order to characterize the electrical properties of cell between two microneedles in the suspension media. The sensing area of mirofluidic chip can be modeled electrically as cell impedance of resistance Rp and cell capacitance Cp in parallel with the impedance contributed by all materials between the two

electrodes, which consist solution resistor Rm in parallel with solution capacitance Cm. Both impedance in series with a pair of electrodes capacitance double layer Cdl. ZT is overall impedance of the measurement system given [21]. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Rm* <sup>+</sup>*Rp* <sup>+</sup> *<sup>j</sup> Rm Rp*(*Cm* <sup>+</sup>*Cp*)

$$\text{of the measurement system given} \quad \text{[21]}.$$

$$Z\_T = \frac{2}{j\alpha C\_{dl}} + \frac{R\_m R\_p}{R\_m + R\_p + j\alpha R\_m R\_p \{C\_m + C\_p\}} \tag{1}$$

where ω is the angular frequency of the electrical signal. As a result, the ZT is changing according the present of cell in the sensing area. The impedance between electrode (microneedle) and electrolyte (solution medium) is our main focus in this work.

#### **1.2 Experimental works**

#### *1.2.1 Cell culture*

In this work, Sacharomycesceresiae cells and microparticle are used as a model for proof of concepts. Sacharomycesceresiae were cultivated in a petri dish containing 10 ml of YPD broth (Yeast extract Peptone Dextrose). The YPD broth contained 1% yeast extract, 2% peptone and 2% glucose. The YPD dishes were incubated at 37°C for 24 hours. The cells were washed with deioinsed (DI) water three times by centrifugation, then they were suspended in sterilized deioinsed water at various dilutions (1:10) concentration. The cells were incubated on agar plates at 37°C for 24 hours for determining the number of cells. The diameter of yeast cells varies from 4 to 7 μm. The number of cells was 1.3 × 109 colony forming units per milliliter (cfu/ ml). The conductivity of DI water is 6 mS/m. The non-fluorescent polystyrene (PS) microbeads with diameter 15 and 9 μm (Polysciences. Inc.) suspended in Phosphatebuffered saline (PBS) solution were diluted to a final concentration of 1000 beads per ml. Polystyrene beads have a known size and electrical properties [22] and have constant impedance across the frequency range used in these experiments.

#### *1.2.2 Device fabrication*

The photolithography technique was utilized to fabricate the microfluidic device. The fabrication begins by designing the masks using layout editor software. The laser lithography system (μPG501, Heidelberg Instruments, Germany) has been used to write the two masks (top and bottom) on the chromium (Cr) masks. Two-step photolithography using SU-82025 negative photoresist (MicroChem, USA) was utilized to fabricate the top layer mold. The first layer has a thickness of 25 μm and was spin coated onto a silicon substrate. After pre-baking, the top layer of Cr mask was place onto the first layer of photoresist for pattern transfer by using a mask aligner (SussMicroTech MA-6), then post-baking with development. Next, the second layer with 60 μm thickness was spin coated on the first photoresist layer and pre-baking. Then, the second layer of photoresist substrate was exposed with the bottom layer Cr mask by UV light. After exposed, the top mold master was obtaining by post-bake and developed process. Meanwhile the bottom mold master with 60 μm thickness was fabricated by following the SU-8 microchannel photolithography technique. PDMS pre-polymers (SYLGARD184A) was thoroughly mixing with curing agents (SYLGARD 184B) in a ratio of 10:1 by weigh for fabricate the PDMS microfluidic chip. The mixing PDMS was poured on an SU-8 mold master (top and bottom mold master) and left for whole night cured at room temperature to obtain the PDMS microfluidic chip. To increase the bonding strength between the top side and bottom side of PDMS microfluidic chip, they were cleaned with Isopropyl alcohol (IPA) and treated by Oxygen plasma (Plasma Etch PE-25) for 25 seconds [23]. The bonding

**105**

**Figure 2.**

*The experimental set-up diagram.*

*Microfluidic Device for Single Cell Impedance Characterization*

are 25, 250 and 31.7 mm, respectively, was utilized.

process of both side PDMS microfluidic chip was completed in less than 2 minutes to prevent loss of Oxygen plasma effectiveness. Finally, the right and left sides of the microchannel chip were cut and leaving a square (60 μm × 120 μm) hole for inserting a microneedle. For measuring electrode, two Tungsten microneedle (Signatone) coated by parylene with tip diameter, shank diameter and length of tungsten needle

The microscope (Olympus Inverted Microscopes IX71) was utilized to monitor the sensing area of microfluidic chip system. The micromanipulator (EB-700, Everbeing) was utilized to insert the two microneedles into microchannel chip through the square hole at right and left side of the chip. For this experiment, the gap between microneedles was fixed at 20 μm. **Figure 2** illustrated the experimental setup of impedance measurement. The two microneedles were connected to impedance analyzer (Hioki IM3570) for input measuring and the result was displayed on the computer. Then, by controlling the syringe pumps (KDS LEGATO 111, KD Scientific, and USA), the 3-ml syringes of the sample solution and yeast concentra-

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

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

to 10<sup>9</sup>

cfu/ml were driven

tion was introduced into microfluidic chip via two tygon flexible tubes.

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

*1.2.4 Impedance measurement procedure*

conductivities 6 and 1.4 S/m respectively.

each seven different concentrations of sample from 102

*1.2.3 Device operation*

process of both side PDMS microfluidic chip was completed in less than 2 minutes to prevent loss of Oxygen plasma effectiveness. Finally, the right and left sides of the microchannel chip were cut and leaving a square (60 μm × 120 μm) hole for inserting a microneedle. For measuring electrode, two Tungsten microneedle (Signatone) coated by parylene with tip diameter, shank diameter and length of tungsten needle are 25, 250 and 31.7 mm, respectively, was utilized.
