Joho Yun

Microelectrical Impedance Spectroscopy for the Differentiation between Normal and Cancerous Human Urothelial Cell Lines: Real-Time Electrical Impedance Measurement at an Optimal Frequency.

Purpose. To distinguish between normal (SV-HUC-1) and cancerous (TCCSUP) human urothelial cell lines using microelectrical impedance spectroscopy (μEIS). Materials and Methods. Two types of μEIS devices were designed and used in combination to measure the impedance of SV-HUC-1 and TCCSUP cells flowing through the channels of the devices. The first device (μEIS-OF) was designed to determine the optimal frequency at which the impedance of two cell lines is most distinguishable. The μEIS-OF trapped the flowing cells and measured their impedance at a frequency ranging from 5 kHz to 1 MHz. The second device (μEIS-RT) was designed for real-time impedance measurement of the cells at the optimal frequency. The impedance was measured instantaneously as the cells passed the sensing electrodes of μEIS-RT. Results. The optimal frequency, which maximized the average difference of the amplitude and phase angle between the two cell lines (p < 0.001), was determined to be 119 kHz. The real-time impedance of the cell lines was measured at 119 kHz; the two cell lines differed significantly in terms of amplitude and phase angle (p < 0.001). Conclusion. The μEIS-RT can discriminate SV-HUC-1 and TCCSUP cells by measuring the impedance at the optimal frequency determined by the μEIS-OF.

Two-Axis Electrostatic Gimbaled Mirror Scanner With Self-Aligned Tilted Stationary Combs

We demonstrate an electrostatic two-axis gimbaled mirror scanner with tilted stationary combs (TSCs). We fabricate the scanner using self-aligned micro-assembly to realize angular offsets between stationary and movable comb electrodes. The TSCs enabled quasi-static operation by generating sufficient scanning angles for the slow axis, whereas in-plane comb electrodes provided torsional oscillation at resonance for the fast axis. The proposed self-alignment method can enhance side stability by minimizing lateral shifts of the electrodes during the micro-assembly. We experimentally verify the fabricated mirror scanner in terms of scanning frequencies and scanning angles for finger-vein authentication. The resonant frequencies and optical-scan angles were 263 Hz and 8.5°, respectively, for the slow axis, and 1959 Hz and 24.6°, respectively, for the fast axis. We obtain high-resolution finger-vein images in transmission mode using the mirror scanner and a near infrared laser, which will possibly improve live finger identification by employing the laser Doppler technique.

Ex vivo characterization of age-associated impedance changes of single vascular endothelial cells using micro electrical impedance spectroscopy with a cell trap

We aimed to characterize aging of single vascular endothelial cells, which are indicators of senescence, using micro electrical impedance spectroscopy (μEIS) for the first time. The proposed μEIS was equipped with two barriers under the membrane actuator near the sensing electrodes, increasing its cell-trapping capability and minimizing the interference between the target cell and subsequent cells. The cell-trapping capability in μEIS with barriers was considerably improved (90%) with a capture time of 5 s or less, compared to μEIS without barriers (30%). Cells were extracted from transgenic zebrafish to minimize an initial discrepancy originating from genetic differences. In order to estimate useful parameters, cytoplasm resistance and membrane capacitance were estimated by fitting an electrical equivalent circuit to the data of ex vivo sensor output. The estimated cytoplasm resistance and membrane capacitance in the younger vascular endothelial cells were 20.16 ± 0.79 kΩ and 17.46 ± 0.76 pF, respectively, whereas those in the older cells were 17.81 ± 0.98 kΩ and 20.08 ± 1.38 pF, respectively. Discrimination of each group with different aging showed statistical significance in terms of cytoplasm resistance (p < 0.001) and membrane capacitance (p < 0.001). Considering both of the sensor and cellular level, the optimal frequency was determined as 1 MHz at which the electrical impedance of each group was clearly discriminated (p < 0.001).

Improvement of Depth Profiling into Biotissues Using Micro Electrical Impedance Spectroscopy on a Needle with Selective Passivation.

A micro electrical impedance spectroscopy (EIS)-on-a-needle for depth profiling (μEoN-DP) with a selective passivation layer (SPL) on a hypodermic needle was recently fabricated to measure the electrical impedance of biotissues along with the penetration depths. The SPL of the μEoN-DP enabled the sensing interdigitated electrodes (IDEs) to contribute predominantly to the measurement by reducing the relative influence of the connection lines on the sensor output. The discrimination capability of the μEoN-DP was verified using phosphate-buffered saline (PBS) at various concentration levels. The resistance and capacitance extracted through curve fitting were similar to those theoretically estimated based on the mixing ratio of PBS and deionized water; the maximum discrepancies were 8.02% and 1.85%, respectively. Depth profiling was conducted using four-layered porcine tissue to verify the effectiveness of the discrimination capability of the μEoN-DP. The magnitude and phase between dissimilar porcine tissues (fat and muscle) were clearly discriminated at the optimal frequency of 1 MHz. Two kinds of simulations, one with SPL and the other with complete passivation layer (CPL), were performed, and it was verified that the SPL was advantageous over CPL in the discrimination of biotissues in terms of sensor output.

Micro electrical impedance spectroscopy on a needle for ex vivo discrimination between human normal and cancer renal tissues

The ex-vivo discrimination between human normal and cancer renal tissues was confirmed using μEoN (micro electrical impedance spectroscopy-on-a-needle) by measuring and comparing the electrical impedances in the frequency domain. To quantify the extent of discrimination between dissimilar tissues and to determine the optimal frequency at which the discrimination capability is at a maximum, discrimination index (DI) was employed for both magnitude and phase. The highest values of DI for the magnitude and phase were 5.15 at 1 MHz and 3.57 at 1 kHz, respectively. The mean magnitude and phase measured at the optimal frequency for normal tissues were 5013.40 ± 94.39 Ω and -68.54 ± 0.72°, respectively; those for cancer tissues were 4165.19 ± 70.32 Ω and -64.10 ± 0.52°, respectively. A statistically significant difference (p< 0.05) between the two tissues was observed at all the investigated frequencies. To extract the electrical properties (resistance and capacitance) of these bio-tissues through curve fitting with experimental results, an equivalent circuit was proposed based on the μEoN structure on the condition that the μEoN was immersed in the bio-tissues. The average and standard deviation of the extracted resistance and capacitance for the normal tissues were 6.22 ± 0.24 kΩ and 280.21 ± 32.25 pF, respectively, and those for the cancer tissues were 5.45 ± 0.22 kΩ and 376.32 ± 34.14 pF, respectively. The electrical impedance was higher in the normal tissues compared with the cancer tissues. The μEoN could clearly discriminate between normal and cancer tissues by comparing the results at the optimal frequency (magnitude and phase) and those of the curve fitting (extracted resistance and capacitance).

An electrostatic two-axis gimbaled mirror scanner with tilted stationary vertical combs fabricated by self-aligned micro-assembly

An electrostatic two-axis gimbaled mirror scanner was demonstrated using tilted stationary vertical combs and self-aligned micro-assembly. Side stability was enhanced and the optical scan angles were 6.8/15.4 degrees on the slow/fast axes, respectively.

MP73-14 CAPABILITY OF ELECTRICAL IMPEDANCE SPECTROSCOPY SENSOR ON A NEEDLE AS A NOVEL TOOL TO ESTIMATE MALIGNANT RENAL TUMOR MARGIN: EX-VIVO DEMARCATION OF TUMOR AND SURGICAL MARGIN

INTRODUCTION AND OBJECTIVES: The combination of the human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir has been a standard regimen used to treat HIV infection. Ritonavir acts as a chemical booster to enhance lopinavir’s activity. Lopinavir has recently been shown to act against cancer by inducing endoplasmic reticulum (ER) stress, and we thought that the combination would kill renal cancer cells by inducing robust ER stress. METHODS: The viability and clonogenicity of renal cancer cells (769-P, 786-O, Caki-2) treated with clinically feasible concentrations of lopinavir (10-40 mM) and/or ritonavir (5-10 mM) were assessed by MTS assay and colony formation assay. Apoptosis was evaluated by annexin-V assay. Cell cycle analysis was done using flow cytometry. Induction of ER stress and the expression of cell-cycle regulators, apoptosis-associated proteins, NOXA, Akt, BCL-2, and survivin were evaluated by western blot analysis. Drug synergism was assessed by the Chou-Talalay method. RESULTS: Lopinavir in combination with ritonavir inhibited renal cancer growth synergistically (combination index <1). The combination also inhibited clonogenic survival of cancer cells significantly (p <0.05). It perturbed the cell cycle by inhibiting the expression of cyclin D1 and cyclin-dependent kinase 4, increasing the cells in the sub-G1 fraction. The combination caused apoptosis synergistically: 10-20 mM lopinavir increased the number of annexin-V positive cells and the expression of cleaved poly(ADP-ribose) polymerase slightly but in combination with 10 mM ritonavir increased both drastically. As expected, the combination induced ER stress evidenced by the increased expression of the ER stress markers glucose-regulated protein 78 and endoplasmic reticulum resident protein 44. Furthermore, increased expression of NOXA confirmed that the combination-induced apoptosis was a result of ER stress. We also found that the combination decreased the expression of the antiapoptotic proteins BCL-2 and survivin by inhibiting the Akt signaling pathway. CONCLUSIONS: The combination of lopinavir and ritonavir induces ER stress and causes renal cancer apoptosis synergistically. Inhibition of the Akt pathway is another important mechanism of its action.

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 µm is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 µm, and the overall width and length of the IDEs are 400 µm and 860 µm, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 µm, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

Evaluation of Electrical Impedance Spectroscopy‐on‐a‐Needle as a Novel Tool to Determine Optimal Surgical Margin in Partial Nephrectomy

A hypodermic needle has been introduced incorporating an electrical impedance spectroscopy (EIS) sensor, called micro-EIS-on-a-needle for depth profiling (μEoN-DP). The μEoN-DP can locate endophytic renal tumors as well as determine tumor margins by detecting the impedance difference between normal and cancer tissues. To evaluate the μEoN-DP as a novel tool to determine the optimal surgical margin during partial nephrectomy (PN), the electrical impedance differences between renal parenchymal tissues and renal cell carcinoma (RCC) tumors are investigated with regard to the distance from the tumors. Optimal frequencies at which the discrimination extent is maximized are suggested based on the discrimination index. The resistance and capacitance of normal and cancer tissues are extracted using electrical equivalent circuit by excluding the influences of other electrical components on the sensor output. The extracted resistance and capacitance of cancer tissues are 37.8% larger and 25.7% smaller than that of normal tissues, respectively. Additionally, high sensitivity and specificity are obtained by using extracted resistance and capacitance, thus implying that the μEoN-DP shows promise as a supplementary tool for PN margin evaluation and decreasing the prevalence of positive surgical margins while maximizing parenchymal preservation.

In-vivo biotissue discrimination using electrochemical impedance spectroscopy on a hypodermic needle with fine interdigitated electrodes

This paper reports the first demonstration of electrochemical impedance spectroscopy-on-a needle (EoN) applied to in-vivo biotissue discrimination. A flexible photomask and photoresist spray coating were employed in the photolithography process to fabricate fine interdigitated electrodes (IDEs) patterns (overall length of 300 μm) at the tip of the round surface of the needle. Prior to main in-vivo experiment, the discrimination capability of the EoN was evaluated at various concentration levels of PBS solution with known electrical properties. Electrical parasitic influence was also investigated as the penetration depth of the IDEs increases. Finally, an in-vivo experiment was conducted to discriminate different kinds of biotissues; the EoN successfully discriminated the fat and muscle tissues of a rat.