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1 J. of the Korean Sensors Society Vol. 19, No. 6 (010) pp DOI : /JSST pissn /eISSN x t v œ w ƒ SAM w CRP Á Á Áy yá A portable surface plasmon resonance sensor system for detection of C-reactive protein using SAM with dimer structure Eun Jung Sin, Eun Jung Joung, Jin Hee Jo, Dong Hwan Hwang, and Young-Soo Sohn Abstract The detection of C-reactive protein(crp) using self-assembled monolayer(sam) was investigated by a portable surface plasmon resonance(spr) sensor system. The CRP is a biomarker for the possible cardiovascular disease. The SAM was formed on gold(au) surface to anchor the monoclonal antibody of CRP(anti-CRP) for detection of CRP. Sequence injection of the anti-crp and bovine serum albumin(bsa) into the sensor system has been carried out immobilize the antibody and to prevent non-specific binding. The portable SPR system has two flow channels: one for the sample measurements and the other for the reference. The output SPR signal was increased with the injection of the anti-crp, BSA and CRP due to binding of the proteins on the sensor chip. The valid output SPR signals was linearly related to the critical range of the CRP concentration. The experimental results showed the feasibility of the portable SPR system with newly developed SAM to diagnose a risk of the future cardiovascular events. Key Words : C-Reactive Protein, surface plasmon resonance, self assembled monolayer, immunosensor 1. w p k ù, š (polymer), g š t ù» œw» w w. ü yw w ƒ y š. d wš w ù z d wš w k w [,3]. k w wý y Quartz Crystal Microbalance e, y w» w (QCM) [4], Neutron Reflection(NR) [5], Fourier Transw [1]. w form Infrared Spectroscopy(FTIR) [6], Field Effect s d ƒ w š Transistor(FET) [7] w ƒ, t w» w ƒ v œ (surface plasmon resonance, SPR) š.» w g ùkù œ ƒ mw, w -w y d w y w w w» w. w SPR w t. w w t w y d w t v w ƒm w œw (Department of Biomedical Engineering, [8]. w w» Catholic University of Daegu) Corresponding author : sohnys@cu.ac.kr (Received : September 16, 010, Revised : October, 010 Accepted : November 3, 010) w ƒ [1,9,10]. w SPR yw 456

2 소형 표면 플라즈몬 공명 센서와 이합체 구조를 가진 SAM을 이용한 CRP 검출 적이며 표면 플라즈몬 공명 현상을 일으키기 쉬운 금 속인 금, 은 등이 있다. 유리 위의 금속 박막, 즉 SPR sensor chip 위에 생분 자를 고정시키기 위한 방안으로는 자기 조립 단분자층 (self-assembled monolayer, SAM)을 이용하는 방법이 대표적이다. SAM은 물질 표면과 SAM의 유기 분자 간의 상호작용에 의해 주어진 표면에 자발적으로 균일 하게 형성되는 단분자막이며 사용 목적에 따라 분자막 의 작용기를 조정할 수 있기 때문에 물질 표면의 기능 화를 위해 널리 사용된다. C-reactive protein(crp) 은 급성 염증성 단백질로, 체내에 염증이 발생하면 간 에서 생성되는 물질이다. CRP는 감염 및 염증성 질환 의 진단 및 치료 후 경과 관찰에 이용할 수 있으며, 최 근에는 CRP의 농도가 높을 때 뇌졸중과 심근경색증의 위험도가 높다는 사실이 알려져 심혈관계 질병의 조기 진단 인자로서 활발히 연구되고 있다. 혈중 1 µg/ml 이하, 1 µg/ml ~ 3 µg/ml, 3 µg/ml 이상의 CRP 농도는 각각 심혈관계 질병 발생률의 낮음, 보통, 높음을 나타 낸다. 또한 10 µg/ml 이상의 혈중 CRP 농도는 급성 염 증성 질환을 진단할 수 있다. 본 연구에서는 소형화된 SPR 센서를 활용하여 sensor chip 위에 이합체 구조를 가진 SAM을 형성하고 이를 이용해 anti-crp를 고정시켜 농도별 CRP를 측정 하였다. 실험 결과를 통해 소형 SPR 센서와 사용된 SAM의 진단용 바이오센서로의 이용 가능성을 조사하 였다. 55 [9] [11] [11,1] Fig. 1. The SPR sensor system including SPRmicro, injection valve, degasser, and peristaltic pump for experiments. [13] [13,14]..1. SPR 센서 실험 방법 본 실험에서는 기존의 SPR 센서에 비해 작은 부피 를 가지는 SPRmicro(K-MAC, Korea)를 사용 하였다. SPRmicro의 부피는 45 mm 140 mm 130 mm로, Fig. 1 에 나타나있다. 이 센서 시스템은 780 nm의 LED 광원 과 D CMOS 검출기를 사용한다. 광원은 Fig. 와 같 이 7.4 (61.3 ~68.7 ) 범위의 입사각으로 금속 박막에 입사되고 D CMOS로 검출된 반사광 중 세기가 가장 약한 반사광의 입사각을 측정하여 공명각을 구한다. 또 한 SPRmicro는 두 개의 채널을 보유하고 있어 하나의 채널을 기준 채널(reference)로 사용할 수 있다. 따라서 주위 환경에 의한 잡음 신호 등 거짓 양성(false-positive) 신호를 제거할 수 있어 정확한 생분자간의 신호 를 얻을 수 있다. Fig. 1에 실제 실험에 사용된 SPRmicro, 시료 주입 밸브, 연동 펌프, 가스 제거기 등을 나 타내었다. o o Fig.. Schematic diagram of SPR sensor... SAM의 형성 실험에 앞서 SPR gold chip을 piranha 용액(황산 (H SO ):과산화수소(H O )=3:1)을 이용하여 세척한 후 DI-water로 잔여물을 제거한 다음 질소(N ) 가스로 건 조 시켰다. 그 후 SAM 용액(1 mm in chloroform, KMAC)에 1시간 동안 반응 시켜 SPR gold chip 표면 에 SAM이 형성 되도록 하였다. 반응 후 chip을 ethanol로 세척한 다음 N 가스를 이용하여 건조 시켰다. 본 실험에서 사용된 SAM은 K-MAC 사에서 제조된 것으로 이합체(dimer) 구조를 가지며, 한 쪽 말단에는 금과 매우 강한 결합을 형성하는 thiol 기를 가지고 있 고 또 다른 말단은 작용기인 N-hydroxysuccinim- o J. Kor. Sensors Soc., Vol. 19, No. 6, 010

3 56 Á Á Áy yá ide(nhs) group. NHS 1 amine w p ƒ» d w [15]..3. Anti-CRP š Anti-CRP CRP p ww w, CRP w» w SPR gold chip t š. SAM x SPR gold chip SPRmicro w z wù w. wù PBS w» w. ƒ PBS w y sx z 150 µg/ml anti-crp(r&d systems, inc. USA) w. Anti-CRP w SPR yƒ ƒw y w PBS w ww anti-crp w. chip t anti- CRP š k z p w» w 100 µg/ml Bovine Serum Albumin(BSA, sigma) w. z chip w û root means square(rms) nm. Fig. 3(b) w» w PBS w SAM anti-crp š k z d w,. x w PBS 1.8 nm rms. Fig. 3(c) anti-crp w 100 µl m w. z BSA w v d 1.94 nm. Fig. 3(d) anti-crp, BSA š CRP¾ w rms 3.10 nm ùkû. ƒ rms ƒw š, w rms mw SAM.4. CRP x mw ƒ CRP d w. CRP(R&D systems, inc. USA) 1, 3, ³ w x q w CRP 5, 10 µg/ml. ƒ 3 x w w t rms ƒ j. w, CRP w w. Anti-CRPƒ š SPR gold chip 3.. CRP d CRP w. CRP w SPR CRP w» w monoclonal yƒ yw y w z ww anti-crp chip t š g w. CRP w» w PBS w š, y ƒ y d w. 3. š 3.1. AFM d Atomic Force Microscopy(AFM) w Fig. 3. AFM images of (a) SAM, (b) SAM/anti-CRP, (c) SAM/anti-CRP/ BSA, (d) SAM/anti-CRP/BSA/ CRP surfaces. w SAM x k SPR gold chip SPRmicro w z anti-crp BSA w. x mw d wš w CRP 1, 3, 5, 10 µg/ml, ƒƒ w ùk ù œ ƒ y w. x SPRmicro ƒ š ƒ y. Fig. 4 ƒ sensor chip t y PBS w» anti-crp Fig. 3 ùkü. SPR w CRP d BSA, š CRP 5 µg/ml w w» w t w v w y ùkü v., e 1 µm 1 µm ƒ y w SPR y y. Fig. 3(a) gold chip SAM x z t y d w» w» k w. roughness y Fig. 5 ùkü. wz 19«6y,

4 x t v œ w ƒ SAM w CRP 57 Fig. 4. Signals from two channels: injection of PBS into reference channel(down) and sequence injection of anti-crp, BSA and 5 µg/ml of CRP into sample channel(up). Fig. 6. Actual signal due to sequence injection of anti- CRP, BSA, 500 ng/ml of PSA and 10 µg/ml of CRP. Fig. 5. Output signal by subtracting signal of reference channel from signal by sequence injection of anti- CRP, BSA, and 5 µg/ml of CRP. CRP z SPR yƒ j ƒw ù, y w SPR yƒ w» PSA w y y 7 RU. w w ùkû. 10 RU w y yƒ y, PSAƒ sensor chip t k w w RU 0.1 o š q w. yƒ z CRP 10 µg/ml œ ƒ y w, SPRmicro w yƒ ƒw, PBS z ùkù 10 RU. ƒ w RU y CRP y 194 RU. z yƒ k 100 mw» SAM š anti-crp w SPR ƒ CRP k w s³ wü. x CRP 3 ww. Chip anti-crp w z chip t x SAM anti-crpƒ š SPR t û anti-crpƒ». Anti-CRP w ƒw y s³ 45 RU. yƒ z p w» w BSA w PBS w. BSA w y s³ 36 RU anti-crp w w, anti-crpƒ chip t sy» š dw. yƒ y w z CRP w y SPR y d w. SPR k» w Prostate- Specific Antigen(PSA, R&D systems, inc. USA) d w Fig. 6 ùkü. SAM x sensor chip SPRmicro k z anti-crp BSA, š PSA 500 ng/ml w. Anti- y w. ƒ CRP w SPR y y s³ Fig. 7. CRP 1 µg/ml s³ 4 RU, CRP yƒ j w. yƒ w PBS 3 µg/ml 86 RU, CRP 5 µg/ml 16 RU, CRP 10 µg/ml w yƒ s w w y w 19 RU j ƒ w. w CRP w SPR. ww ù t sy z y y anti-crp w y 459 J. Kor. Sensors Soc., Vol. 19, No. 6, 010

5 58 Á Á Áy yá SAM w w š k SPRmicroƒ x y x w» w CRP w ww y w. 010 ( w» ) w w» (No ). Fig. 7. Mean values of SPR signals caused by binding of CRP with various concentrations: The concentrations of CRP were 1, 3, 5, and 10 µg/m. š x [1] H. Chen, J. Huang, J. Lee, S. Hwang, and K. Koh, ùkû. w w y Surface plasmon resonance spectroscopic characterizatin of antibody orientation and activity on the j SPR yƒ yw, anti-crp ( calixarene monolayer, Sens. Actuators B, vol. 147, 150 kda) CRP ( 3 kda) w jš pp , 010. x anti-crp (150 µg/ml)ƒ CRP (1, 3, 5, 10 µg/ml)» q. w mw Fig. 7 ùkù CRP 1 µg/ml 10 µg/ml SPR y y x ƒw. x mw curve fitting y % µg/ml RU ùkû. 1 µg/ml 10 µg/ml x CRP x y x y w e. mw w ƒ SAM x SPR ƒ CRP w y w. 4. xy SPR SAM w t CRP w. x CRP 1, 3, 5, 10 µg/ml, x ƒ w. SPRmicro wù» w y w w w yw y. x w y» y wü š, ƒ y w SPR yƒ ƒw y w. CRP w y 1 µg/ml 10 µg/ml x ƒ w. mw w ƒ [] P. J. Conroy, S. Hearty, P. Leonard, and R. J. O Kennedy, Antibody production, design and use for biosensor-based applications, Semin. Cell Dev. Biol., vol. 0, pp. 10-6, 009. [3] R. M. Lequin, Enzyme Immunoassay(EIA) / Enzyme- Linked Immunosorbent Assay, Clin. Chem., vol. 51, pp , 005. [4] R. Hao, D. Wang, X. Zhang, G. Zuo, H. Wei, R. Yang, Z. Zhang, Z. Cheng, Y. Guo, Z. Cui, and Y. Zhou, Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor, Biosens. Bioelectron, vol. 4, pp , 009. [5] H. Xu, X. Zhao, C. Grant, J. R. Lu, D. E. Williams, and J. Penfold, Orientation of a monoclonal antibody adsorbed at the solid/solution interface: a combined study using atomic force microscopy and neutron reflectivity, Langmuir, vol., pp , 006. [6] M. Salmain, N. Fischer-Durand, and C.-M. Pradier, "Infrared optical immunosensor: Application to the measurement of the herbicide atrazine, Anal. Biochem. vol. 373, pp , 008. [7] Y.-S. Sohn, C.-K. Kim, and S.-Y. Choi, Characteristics of a label-less electrochemical immunosensor based on a field-effect transistor for the detection of a biomarker in urine, Sens. Lett. vol. 7, pp , 009. [8], x, x, x, Cycilic olefin copolymer(coc) s v w Ÿ» t v œ (SPR), wz 19«6y,

6 x t v œ w ƒ SAM w CRP 59 wz, 17«, 5y, pp , 008. [9] R. B. M. Schasfoort and A. J. Tudos, Handbook of surface plasmon resonance, Royal Society of Chemistry, London, 008. [10] z,», Ÿ y t v œ w Ÿ t ù x, wz, 17«, y, pp , 008. [11] S. K. Arya, P. R. Solanki, M. DattaS and B. D. Mal- hotra, Recent advances in self-assembled monolayers based biomolecular electronic devices, Biosens. Bioelectron, vol. 4, pp , 009. [1] K.-Y. Park, Y.-S. Sohn, C.-K. Kim, H.-S. Kim, Y.- S. Bae, and S.-Y. Choi, Development of FET-type albumin sensor for diagnosing nephritis, Biosens. Bioelectron, vol. 3, pp , 008. [13] M. Hamer, Y. Chida, and E. Stamataki, Utility of C-reactive protein for cardiovascular risk stratification across three age groups in subjects without existing cardiovascular diseases, Am. J. Cardiol., vol. 104, pp , 009. [14] H. Y. Tsai, C. F. Hsu, I. W. Chiu, and C. B. Fuh, Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles, Anal. Chem., vol. 79, pp , 007. [15] H.-M. Cho, D.-H. Jang, S.-K. Lee, S.-J. Ku, H.-C. Kim, K.-S. Yu, and J.-W. Lee, Characterization of novel self assembled materials(sam) for surface modification of sensor chip, Biochip J., vol., pp , ~ 010 ƒm w 010 ~ x ƒm w œw 007 ~ x ƒm w 006 ~ x ƒm w y y 007 ~ x ƒm w wz 19«, y, p. 148, 461 J. Kor. Sensors Soc., Vol. 19, No. 6, 010

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