Sensors and Actuators B 117 (2006) 332338

Label-free protein assay with site-directly immobilized antibody using self-actuating PZT cantilever
Ghi Yuun Kang a,b , Ga Young Han a , Ji Yoon Kang a , Il-Hoon Cho c , Hyung-Ho Park b , Se-Hwan Paek c , Tae Song Kim a,
a

Microsystem Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea b Department of Ceramic Engineering, Yonsei University, Seoul 120-749, Republic of Korea c Program for Bio-Microsystem Technology, Korea University, Seoul 136-701, Republic of Korea Received 5 October 2005; accepted 7 November 2005 Available online 10 January 2006

Abstract This paper reports a method to improve the limit of the label-free detection of myoglobin using site-directed immobilization on the surface of self-actuating PZT cantilever sensor. The resonant frequency change of cantilever was measured electrically using piezoelectricity when antibodyantigen interaction occurred. Site-directed antibody was biotinylated after reducing the binding region to hinge region of randomly biotinylated antibody, which increased the number of effective binding sites. Investigating the effect of site-directed antibody on the cantilever sensor revealed that this protocol was able to enhance the performance of analytical sensitivity about 10 times with the modication of sensing layer. The experimental results demonstrated this biosensor can be used for diagnosis of acute myocardial infarction in clinical range of tens of ng/ml. 2005 Elsevier B.V. All rights reserved.
Keywords: PZT microcantilever; Electrical detection; Site-directly immobilized antibody; Myoglobin

1. Introduction Microcantilever biosensor is attracting intense attention because of its label-free detection and high sensitivity [13]. Since it transduces the changes of surface condition into electrical or optical signals without labeling of molecule, it is thought to be one of promising label-free detection methods such as surface plasmon resonance (SPR), resonant waveguide grating or quartz crystal microbalance (QCM) [5]. Nanomechanical biosensors with high sensitivity have been successfully applied to many kinds of assays such as specic molecular interactions [46], cell adhesion [7] and chemical gases [810]. The most well-known example of microcantilever sensor is static type sensor, which is demonstrated by IBM Zurich [11] and UC Berkeley [12]. They were silicon-based cantilevers and it measured the tip displacement of cantilever caused by the surface stress of target DNA or protein molecules, which are

Corresponding author. Tel.: +82 2 958 5564; fax: +82 2 958 6910. E-mail address: tskim@kist.re.kr (T.S. Kim).

specically bound to their receptors immobilized on the surface of the cantilever [1315]. However, due to the nanometer range of tip movement, the measurement requires expensive and bulky laser and photodiode system, and it is very sensitive to the environmental disturbance. Other than static bending cantilever, dynamic cantilever sensor which measure the mechanical resonant frequency of cantilever is robust against external vibration since it is cantilevers own mechanical property. The dynamic type sensor can detect the mass of bound biomolecules as well as surface stress [16,17]. Hence, as long as the bio agent stays on the surface of the cantilever, the resonance frequency after adsorption remains unchanged since there is no interaction of biomolecules in dry environment. However, most dynamic cantilever sensors require external actuator for vibration and complex optical measuring system to detect resonance frequency. Therefore, we developed self-actuating cantilever for the electrical detection of vibration with monolithic actuator. When compared to thermal or magnetic actuation, piezoelectric cantilever has its own merits because it can simultaneously detect the mechanical resonance electrically and drive the cantilever. We used a

0925-4005/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.11.011

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Pb(Zr0.52 Ti0.48 )O3 (PZT) as a piezoelectric material in monolithic microcantilever [6]. Biosensor usually consists of sensing layer, transducer and electronic circuits. To achieve low detection limit, most research groups are working on the improvement of transducing mechanism, however, sensing layer is also important factor in enhancing sensor performance. This paper focuses on sensing layer of microcantilever and investigated its effect on the change of surface property. The sensing layer of this paper is myoglobin antibody and the resonance frequency was changed by the antibodyantigen reaction. Myoglobin is one of the most reliable clinical makers for diagnosis of acute cardiac infarction and its clinical range is 2.70510 ng/ml [1820]. Detection of myoglobin concentration of a patient is also important in monitoring the progression of disease in patients and the effect of treatment. Moreover, the electrical detection of resonant frequency shift of cantilever sensor is able to realize the point-of-care testing of acute myocardial infarction since electrical sensing system is small and fast enough for the emergency case. When developing immunosensors, the problem of sensing layer is the efciency of binding site of antibody. The antibodies are adsorbed on the sensor surface or covalently coupled via amino and carboxyl groups and all the binding sites are not exposed to react since antibody has many functional groups to be bound to the surface. The random orientation of the antibodies limits the proportion of available binding sites in spite that the surface is fully saturated with antigen. Hence, we control the surface-binding site of antibody and control the orientation of antibody to increase the number of antibodies to interact with antigen. This site-directed immobilization leads to high activity of sensing layer. Although we showed that the site-directed antibody enhanced the sensitivity using optical method [2123], no report was published about the effect of the site-directed antibody on the microcantilever sensor. We adopted the site-directed immobilization to the surface of cantilever and investigated its effect on the electrical sensing of resonance frequency.

2. Experimental procedure 2.1. Fabrication of a PZT thin lm microcantilever and electrical measurement Nanomechanical PZT cantilevers were fabricated in vesteps as shown in Fig. 1. First, the substrate of PZT capacitor was 100 mm p-doped Si(1 0 0) wafer covered with 1.2 mthick low-stress silicon nitride (SiNx ), which was deposited by low-pressure chemical vapor deposition (LPCVD). The bottom electrode layer was deposited by sputtering thin tantalum adhesion layer followed by a platinum layer with thickness of 150 nm. The PZT lm 0.5 m thick was spin-coated by diolbased solgel route. For the metalferroelectricmetal capacitor structure, we used dc sputtering to deposit a Pt layer for the top electrode. Top electrode and PZT layer were patterned by inductively coupled plasma (ICP) etching followed by ion milling for the patterning of the Pt bottom electrode. After etching the rear SiNx window with reactive ion etching (RIE), the bulk silicon was wet etched with KOH silicon etchant. Finally, the cantilever was formed by the reactive ion etching of SiNx layer. After fabricating the PZT cantilever, 0.2 m thick silicone oxide was deposited for electrical and chemical passivation layer. Fig. 2 shows SEM photographs of the PZT cantilever arrays designed for self-sensing with dimensions of 100 m 300 m (width length). The cantilever was 2.08 m thick in total and it composed of SiO2 /SiNx /Ta/Pt/PZT/Pt/SiO2 multilayer. Due to their low processing temperature condition, this piezoelectric thin lm cantilever is compatible with CMOS electronics for further electrical signal processing and actuation. When measuring the resonant frequency, sinusoidal waveform with the dc bias voltage of 0.25 V was applied at the top electrode. The rst resonant frequency of cantilever was found at the frequency of the maximum deection after frequency sweeping actuation. At a driving amplitude of 1 Vpp, the frequency response exhib-

Fig. 1. (ae) The fabrication ows of self-actuating PZT microcantilever.

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6.0, and was treated with variable concentrations of MEA (1100 mM after dilution, Mercaptoethylamine, Sigma Co., USA) as a reducing agent. After allowing the reaction to run for 90 min at room temperature, the excess MEA was removed by size-exclusion chromatography on a Sephadex G-15 gel column. The degree of the reduced state of immunoglobulin was veried by sodium dodecyl sulphate (SDS)-polyacrlamide gel electrophoresis (PAGE) using 12% gel. The samples were treated with a 60 mM TrisHCl buffer of pH 6.8, which consisted of 25% glycerol, 2% SDS and 0.1% bromophenol blue. After loading the samples, the separation was run under 100 V and 40 mA for 90 min. The gel was stained with a 0.1% Coomassie Blue R-250 solution containing 45% methanol and 10% acetic acid for 1 h, and then, the gel was destained. Using an optimal concentration of MEA (50 mM), the reduced antibody was immediately reacted with 20 molar excess of biotin-BMCC (1-biotinamido-4-(4 -[maleimidoethylcyclohexane]-carboxamido) butane, Pierce Co., USA) that had been dissolved in dimethyl sulfoxide for 2 h at room temperature. Unreacted biotin molecules were removed by dialysis and subsequently by size-exclusion chromatography. 2.3. Immunoassay of the site-directed and randomly immobilized antibodies To investigate the performance enhancement of site-directed biotinylated antibody, randomly biotinylated antibody immobilization was prepared as a negative control. The schemes of immunoassays on streptavidin-coated surfaces are depicted in Fig. 4. The randomly biotinylated antibody as the control was synthesized by coupling 20 molar excess of N-hydroxysuccinimidyl (NHS)-biotin to the intact antibody. The excess biotin was then removed by dialysis and gel ltration. For immobilization of the antibodies, streptavidin (10 g/ml) dissolved in phosphate buffered saline (PBS) solution was coated on the surfaces of the cantilever that were already treated with piranha solution (H2 O2 :H2 SO4 = 1:4). The cantilever devices were incubated within a closed box, and 100% humidity was maintained for 1 h at 37 C. These same conditions were also used for incubations in the following steps that are mentioned. After washing, the residual surface was blocked with bovine serum albumin (BSA), and

Fig. 2. SEM photographs of the microfabricated self-actuating PZT microcantilever with dimensions (W L) of 100 m 300 m (a) cantilever of three array and (b) single cantilever.

ited Lorentzian characteristics without severe electromechanical non-linearity. The electrical measuring was veried through the optical measurement. The equipment was heterodyne laser Doppler vibrometer (MLD211D, Neo ark Co., Japan). The change of resonant frequency due to the antibodyantigen interaction was electrically detected for the various concentration of interacted myoglobin antigen. Fig. 3 shows schematic diagram of electrical measurement system using impedance analyzer (HP4294A, Agilent Technologies Co., USA). 2.2. Site-directed biotinylation of immunoglobulin The monoclonal antibody to myoglobin (Fitzerald Co., USA) was rst dialyzed against a 100 mM phosphate buffer, pH

Fig. 3. Schematic diagram of electrical measurement system using impedance analyzer.

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Fig. 4. Schematic diagram of conceivable conguration formed in the respective immunoassay system with different capture antibodies. One was the antibody biotinylated at the dened hinge region (upper) while the other at the random position (lower).

each antibody (10 g/ml), which was dissolved in BSA containing 0.1% Tween 20 (BSA-TW), was immobilized on separate streptavidin-coated surfaces of the PZT cantilever. Myoglobin monoclonal antibody (10 g/ml) was allowed to react in each device. Streptavidin was used as a linker for the immobilization of two different biotinylated myoglobin antibodies on Au surface of the cantilever. Then, blocking was performed using BSA in order to prevent non-specic binding on the transducer surface. After myoglobin antigen was added onto myoglobin antibody, cantilever was washed out by PBS solution and then dried with nitrogen gas. Resonant frequency shift was measured under ambient atmosphere of controlled humidity at RH 80% and temperature at 37 C. 3. Results and discussions 3.1. Experimental analysis of quantitative myoglobin antigen using uorescence image Using uorescence image of laser confocal scanner (ScanArray Gx, Perkin-Elmer Co., USA), we examined the myoglobin antigenantibody interaction as a function of the myoglobin antigen concentration. Myoglobin antigen was tagged by Cy-5 and on the cantilever was functionalized by site-directed myoglobin antibody. Fig. 5 shows the uorescence images when the interacted myoglobin antigen concentrations were (a) 1 g/ml, (b) 100 ng/ml, (c) 10 ng/ml and (d) 1 ng/ml, and (e) negative control (1 mg/ml of BSA). The uorescent intensity indicates that the myoglobin antigenantibody was reasonably proportional to the concentration of the myoglobin antigen biomarker. The negative control result means that the myoglobin antibody immobilized surface is selective to only myglobin antigen. As shown in Fig. 5e, the uorescence image reveals negligible nonspecic binding between the myoglobin antibody and the BSA protein, which implies the high specicity of the antibody for myoglobin protein against other proteins. We can see the distinct changes in confocal intensity as a function of concentration of myoglobin antigen. The uorescence images also indicate the presence of myoglobin antigen surface only on the Au surface. This suggests that the change in the resonant frequency occurred only due to the antibodyantigen interaction.

3.2. Quantitative electric detection with changes in resonant frequency We analyzed the minimum detectable concentration of the myoglobin antigen using cantilever sensor. After the immobilization of site-directed and random myoglobin antibody on Aucoated cantilever surface, we compared the resonance changes in the clinical range of myoglobin concentration. The reso nant frequency of cantilever (f = 1/2 k/m ) is related to the spring constant (k) and the effective mass (m* ), where the effective mass (m* ) of the rectangular cantilever is 0.236 m. On the assumption that the beam spring constant (k) remains constant during mass loading, and the mass was uniformly deposited, then the loading mass m is estimated in the following equation, [7]:
2 2 f1 f2 m = 2 m f1

(1)

where f2 is the resonance frequency after the mass loading and f1 is the resonance frequency before the mass loading. However, during the AbAg interaction the compressive surface stress reportedly occurs on the functionalized side of the cantilevers due to repulsive electrostatic/steric intermolecular interactions and the changes in hydrophobicity of surface [11]. The resonant frequency shift of PZT cantilever occurs due to both the change of spring constant and mass loading in protein adsorption [17]. The fabrication process must therefore be controlled to such an extent that the mechanical properties of the PZT nanomechanical cantilevers must almost be identical to measure the subtle change of the surface. All the resonant frequency of the cantilevers used in experiments reveals a variation of less than 2%. The error, which could be generated from both non-specic binding and the conditions of the measuring environment, can be compensated by extracting the differential frequency change in the negative controlled cantilever. Experimental results in Fig. 6 revealed that normalized frequency shift was proportional to the myoglobin antigen concentrations with both antibodies. The specic binding of myglobin antigen caused normalized frequency change of 10.5 103 for the site-directly immobilized cantilever and 5.9 103 for randomly immobilized cantilever when the myoglobin antigen

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Fig. 5. The uorescence scanner images as a function of the interacted myoglobin antigen concentrations with (a) 1 g/ml, (b) 100 ng/ml, (c) 10 ng/ml and (d) 1 ng/ml, and as a negative control, with (e) 1 mg/ml of BSA on functionalized cantilever with site-directly immobilized myoglobin antibody.

concentration was 10 g/ml. The sensitivity for the site-directly biotinylated antibody was enhanced approximately twice compared to that of the randomly biotinylated antibody. As shown in Fig. 6, we achieved typical curves of resonant frequency change as a function of the myoglobin antigen concentration and these curves are similar to that of the ELISA obtained by Cho et al. [23]. Considering the variation of sensing result, the estimated limit of detection of site-directed antibody is around 1 ng/ml, while that of random antibody is 10 ng/ml. Hence,

the effective sensing layer enhanced the analytical sensitivity about 10 times and this enabled the sensor to be used in clinical diagnosis. It is interesting that the slope of resonant frequency changes as a function of myoglobin antigen concentration reveals same slope between the different antibodies, while the magnitude of resonant frequency changes increases for site-directed biotinylated antibody. We believe that the increase of resonant frequency for site-directly biotinylated antibody results in the

Fig. 6. Normalized rst resonant frequency shifts as a function of myoglobin antigen concentration on antibody of randomly and site-directly biotinylated immobilized surface with the dimension of 100 m 300 m cantilever. The myoglobin antibody concentration is xed at 10 g/ml.

Fig. 7. Normalized rst resonant frequency shifts as a function of concentration on antibody of randomly and site-directly biotinylated immobilized surface with the dimension of 100 m 300 m cantilever. The myoglobin antigen concentration is xed at 1 g/ml.

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increase of minimum detectable sensitivity. The results clearly indicate that the immobilization method of the cantilever affects its detection sensitivity. Fig. 7 shows the experimental results of the normalized rst resonant frequency changes as a function of the myoglobin antibody concentration which myoglobin antigen concentration was xed at 1 g/ml. The result clearly indicates that the sitedirectly antibody immobilization method is effective at high antibody concentration region. Hence, the antibody concentration was selected as 10 g/ml for our immobilization process. The specic binding of myglobin antigen to its antibody caused a normalized resonant frequency change of 11.36 103 for the site-directly immobilized cantilever and 8.05 103 for randomly immobilized cantilever. 4. Conclusions In this study, we suggested a method of antibody immobilization to improve the sensitivity of PZT cantilever sensor. The sensing signal was electrically measured under controlled ambient environment for label-free detection. The experimental analysis suggests that the resonant frequency change of the site-directed immobilized myoglobin antibody was higher that of randomly immobilized antibody. This result proposed that the site-directly biotinylated method effectively decrease the minimum analytical sensitivity of the cantilever biosensor by more than 1 order. The appropriate combination of the PZT nanomechanical cantilever and the effective protein immobilization process will provide a simple, label-free electric detection system for portable diagnostic system. Acknowledgements This research has been supported by the Intelligent Microsystem Center (IMC; http://www.microsystem.re.kr), which carries out one of the 21st centurys Frontier R&D Projects sponsored by the Korea Ministry Of Commerce, Industry and Energy and a KIST GRANT Program sponsored by KIST. References
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Biographies
Ghi Yuun Kang received his Master degree in material science and engineering from Chungnam National University Korea in 1994. From 2000 to 2005 he joined KIST and Yonsei University, Korea as PhD student. His research interests include Bio-MEMS, nanomechanics, integrated micro/nanodevice, piezoresistive sensor and piezoelectric material. Ga Young Han received the BS in chemical engineering, in 2003, from Sungkyunkwan University, Suwon. She is currently working toward the MS

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G.Y. Kang et al. / Sensors and Actuators B 117 (2006) 332338 of Yonsei University in Korea. His researches focus on the preparation and characterization of Pb-based ferroelectric thin lms and the interface with electrode materials for using them as capacitor, sensor, gate materials; GaAs surface and interface for using it in MOS; meso-porous materials including SiO2 aerogel lm for applying it to intermetal dielectric of ULSI devices; DLC thin lm for applying it to FED and hard coating; organic thin lms containing dispersed nano-particles for applying them to EL devices. Se-Hwan Paek obtained his PhD from University of Michigan, USA, in 1991. He is currently professor of Department of Biotechnology and Bioinformatics, Korea University, Korea. His research interests surface modication, selfassembly monolayer, enzyme assay. Tae Song Kim obtained his PhD from the Korea Advanced of Scientic Institute and Technology (KAIST), Daejeon, Korea in 1993. He is currently a Principal Research Scientist at KIST and the Director of Intelligent Microsystem Center (IMC), Korea. He was Postdoctoral Associate studying microelectromechanical systems (MEMS) devices at Department of Electrical and Computer Science Engineering, University of Minenesota, from 1997 to 1998. He was a director of the Microsystem Research Center at KIST from 2001 to 2004. His research interests include Bio-MEMS, biosensor, protein chip, microuidics, integrated micro/nanodevice and piezoelectric MEMS.

degree in chemical and biological engineering from Korea University, Seoul. She is student researcher with the Korea Institute of Science and Technology, Seoul. Her research interests include surface modication and biomolecule immobilization on biosensor/nanodevice. Ji Yoon Kang obtained his PhD in mechanical engineering from Seoul National University, Korea, in 1997. After working as a member of research staff at Samsung Advanced Institute of Science and Technology, he has been a senior research scientist at KIST since 2001. He was a post-doctoral associate studying plastic biochip at University of Cincinnati from 2002 to 2003. He is currently interested in microuidic biochemical assay system, nanobiosensors and cell-based assay lab-on-a-chip. Il-Hoon Cho obtained his Master degree in program for biomicrosystem technology, Korea University, Korea, in 2004. He is currently PhD student in Program for biomicrosystem technology, Korea University, Korea. His research interests surface modication, self-assembly monolayer, enzyme assay. Hyung-Ho Park received his PhD in materials science in 1988 from the University of Bordeaux I in France. After 1 year of post-doc at chemistry laboratory of CNRS Bordeaux, he joined Electronics & Telecommunications Research Institute (ETRI) in Korea in the eld of processing and characterization of semiconductors. Since 1995 he is Professor of ceramic department