인플루엔자 바이러스 검출을 위한 종이 기반 neuraminidase 효소 활성 평가 센서 개발

황철환; 정성근; 박한규; 이창수; 김윤곤; Cheol-hwan Hwang; Seong-Geun Jeong; Han-Kyu Park; Chang-Soo Lee; Yun-Gon Kim

Korean Chemical Engineering Research, Vol.54, No.3, 380-386, June, 2016

DOI10.9713/kcer.2016.54.3.380 Export Citation

인플루엔자 바이러스 검출을 위한 종이 기반 neuraminidase 효소 활성 평가 센서 개발

Paper-Based Neuraminidase Assay Sensor for Detection of Influenza Viruses

황철환, 정성근¹, 박한규, 이창수^{1, †}, 김윤곤^†

숭실대학교 화학공학과, 06978 서울특별시 동작구 상도로 369
¹충남대학교 화학공학과, 34134 대전광역시 유성구 대학로 99

Cheol-hwan Hwang, Seong-Geun Jeong¹, Han-Kyu Park, Chang-Soo Lee^{1, †}, and Yun-Gon Kim^†

Department of Chemical Engineering, Soongsil University, 369, Sangdo-ro, Dongjak-gu, Seoul, 06978, Korea
¹Department of Chemical Engineering, Chungnam National University, 99, Daehak-ro, Yuseong-gu, Daejeon, 34134, Korea

E-mail:,

초록

본 연구에서는 인플루엔자 바이러스 표면에 존재하는 neuraminidase 효소의 활성을 평가 할 수있는 종이칩 기반의 분석 시스템을 구축하였다. 종이칩의 장점을 살려 분석 전문가와 장비 없이 현장 진단(Point-of-care)이 가능하도록 X-Neu5Ac 기질을 이용한 비색분석법을 통해 시료 내 neuraminidase 효소의 존재를 정량적으로 확인 할 수 있도록 설계 및 제작하였다. Neuraminidase 효소의 활성을 확인할 수 있는 종이칩 센서(Paper-based neuraminidase assay sensor; PNAS) 성능 실험 결과 neuraminidase를 0.004 U/mL 농도부터 검출 가능하였으며, 인간 혈청에 각기 다른 농도로 존재하는 neuraminidase 효소의 양을 활성 평가를 통해 정량적으로 검출할 수 있음을 입증하였다(R2 > 0.99). 또한, 보관기간에 따른 종이칩의 안정성 평가 결과 빛이 차단 된 4 ℃ 환경에서 보관 시 70일까지 초기 성능이 안정하게 유지됨을 확인하였다. 마지막으로, PNAS 상에서 효소 반응의 신뢰성 평가를 위해 미카엘리스-멘텐 동역학 (Michaelis-Menten kinetics)을 적용하여 X-Neu5Ac 기질에 대한 neuraminidase의 동역학 분석 결과 Km 값은 8.327×10-3 M으로 확인되었으며, 이 값은 용액상에서의 효소 반응 속도 계산으로부터 산출된 값과(Km=0.89×10-3 M) 근사한 수치임을 확인하였다. 본 연구로부터 개발된 종이칩 기반의 neuraminidase 효소 활성 평가 시스템은 인플루엔자 바이러스의 신속하고 안전한 검출에 다양하게 응용 될 수 있을 것으로 생각된다.

In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity (R2 > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the 4 ℃. Finally, we demonstrated whether the Michaelis-Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions (Km=8.327×10-3 M), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.

Keywords:Influenza virus;Neuraminidase;Paper chip;Enzyme kinetics;X-Neu5Ac;Colorimetric assay

[References]

Harper SA, Fukuda K, Uyeki TM, Cox NJ, Bridges CB, MMWR Prev Control, 53, 1 (2004)
Katagiri S, Ohizumi A, Homma M, J. Infect. Dis., 148(1), 51 (1983)
Matsuzaki Y, Katsushima N, Nagai Y, Shoji M, Itagaki T, Sakamoto M, Kitaoka S, Mizuta K, Nishimura H, J. Infect. Dis., 193(9), 1229 (2006)
De Jong J, Rimmelzwaan G, Fouchier R, Osterhaus A, J. Infect. Dis., 40(3), 218 (2000)
Das K, Aramini JM, Ma LC, Krug RM, Arnold E, Nat. Struct. Mol. Biol., 17(5), 530 (2010)
Moscona A, N. Engl. J. Med., 353(13), 1363 (2005)
Wiley DC, Skehel JJ, Annu. Rev. Biochem., 56(1), 365 (1987)
Horimoto T, Kawaoka Y, Nat Rev Micro, 3(8), 591 (2005)
Benton DJ, Martin SR, Wharton SA, McCauley JW, J. Biol. Chem., 290(10), 6516 (2015)
Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD, J. Virol., 78(22), 12665 (2004)
Amano Y, Cheng Q, Anal. Bioanal. Chem., 381(1), 156 (2005)
Spackman E, Senne DA, Myers T, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL, J. Clin. Microbiol., 40(9), 3256 (2002)
Voller A, Bartlett A, Bidwell D, Clark M, Adams A, J. Gen. Virol., 33(1), 165 (1976)
Schafer W, Pister L, Hunsmann G, Moennig V, Nature, 245(142), 75 (1973)
Bassin RH, Tuttle N, Fischinger PJ, Nature, 229, 564 (1971)
Yager P, Edwards T, Fu E, Helton K, Nelson K, Tam MR, Weigl BH, Nature, 442(7101), 412 (2006)
Yager P, Domingo GJ, Gerdes J, Annu. Rev. Biomed. Eng., 10(1), 107 (2008)
Gervais L, de Rooij N, Delamarche E, Adv. Mater., 23(24), H151 (2011)
Boehm DA, Gottlieb PA, Hua SZ, Sens. Actuators B-Chem., 126(2), 508 (2007)
Fujii T, Microelectron. Eng., 61, 907 (2002)
Plecis A, Chen Y, Microelectron. Eng., 84(5), 1265 (2007)
Huh YS, Choi BG, Hong WH, Korean Chem. Eng. Res., 50(4), 738 (2012)
Huh YS, Jeon SJ, Lee EZ, Park HS, Hong WH, Korean J. Chem. Eng., 28(3), 633 (2011)
Parolo C, Merkoci A, Chem. Soc. Rev., 42(2), 450 (2013)
Gomez FA, Bioanalysis, 6(21), 2911 (2014)
Martinez AW, Phillips ST, Whitesides GM, Carrilho E, Anal. Chem., 82(1), 3 (2010)
Jokerst JC, Adkins JA, Bisha B, Mentele MM, Goodridge LD, Henry CS, Anal. Chem., 84(6), 2900 (2012)
Carrilho E, Martinez AW, Whitesides GM, Anal. Chem., 81(16), 7091 (2009)
Martinez AW, Phillips ST, Wiley BJ, Gupta M, Whitesides GM, Lab Chip, 8(12), 2146 (2008)
Fujii I, Iwabuchi Y, Teshima T, Shiba T, Kikuchi M, Biorg. Med. Chem., 1(2), 147 (1993)
Vella SJ, Beattie P, Cademartiri R, Laromaine A, Martinez AW, Phillips ST, Mirica KA, Whitesides GM, Anal. Chem., 84(6), 2883 (2012)
Cheng CM, Martinez AW, Gong J, Mace CR, Phillips ST, Carrilho E, Mirica KA, Whitesides GM, Angew. Chem.-Int. Edit., 49(28), 4771 (2010)
Saito M, Hagita H, Iwabuchi Y, Fujii I, Ikeda K, Ito M, Histochem. Cell Biol., 117(5), 453 (2002)
Minami A, Otsubo T, Ieno D, Ikeda K, Kanazawa H, Shimizu K, Ohata K, Yokochi T, Horii Y, Fukumoto H, PLoS One, 9(1), e81941 (2014)
Michaelis L, Menten ML, Biochem. z, 49(333-369), 352 (1913)
Fanjul-Bolado P, Gonzalez-Garcia MB, Costa-Garcia A, Anal. Bioanal. Chem., 386(6), 1849 (2006)

[Referenced By]

[1] Harper SA, Fukuda K, Uyeki TM, Cox NJ, Bridges CB, MMWR Prev Control, 53, 1 (2004)

[2] Katagiri S, Ohizumi A, Homma M, J. Infect. Dis., 148(1), 51 (1983)

[3] Matsuzaki Y, Katsushima N, Nagai Y, Shoji M, Itagaki T, Sakamoto M, Kitaoka S, Mizuta K, Nishimura H, J. Infect. Dis., 193(9), 1229 (2006)

[4] De Jong J, Rimmelzwaan G, Fouchier R, Osterhaus A, J. Infect. Dis., 40(3), 218 (2000)

[5] Das K, Aramini JM, Ma LC, Krug RM, Arnold E, Nat. Struct. Mol. Biol., 17(5), 530 (2010)

[6] Moscona A, N. Engl. J. Med., 353(13), 1363 (2005)

[7] Wiley DC, Skehel JJ, Annu. Rev. Biochem., 56(1), 365 (1987)

[8] Horimoto T, Kawaoka Y, Nat Rev Micro, 3(8), 591 (2005)

[9] Benton DJ, Martin SR, Wharton SA, McCauley JW, J. Biol. Chem., 290(10), 6516 (2015)

[10] Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD, J. Virol., 78(22), 12665 (2004)

[11] Amano Y, Cheng Q, Anal. Bioanal. Chem., 381(1), 156 (2005)

[12] Spackman E, Senne DA, Myers T, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL, J. Clin. Microbiol., 40(9), 3256 (2002)

[13] Voller A, Bartlett A, Bidwell D, Clark M, Adams A, J. Gen. Virol., 33(1), 165 (1976)

[14] Schafer W, Pister L, Hunsmann G, Moennig V, Nature, 245(142), 75 (1973)

[15] Bassin RH, Tuttle N, Fischinger PJ, Nature, 229, 564 (1971)

[16] Yager P, Edwards T, Fu E, Helton K, Nelson K, Tam MR, Weigl BH, Nature, 442(7101), 412 (2006)

[17] Yager P, Domingo GJ, Gerdes J, Annu. Rev. Biomed. Eng., 10(1), 107 (2008)

[18] Gervais L, de Rooij N, Delamarche E, Adv. Mater., 23(24), H151 (2011)

[19] Boehm DA, Gottlieb PA, Hua SZ, Sens. Actuators B-Chem., 126(2), 508 (2007)

[20] Fujii T, Microelectron. Eng., 61, 907 (2002)

[21] Plecis A, Chen Y, Microelectron. Eng., 84(5), 1265 (2007)

[22] Huh YS, Choi BG, Hong WH, Korean Chem. Eng. Res., 50(4), 738 (2012)

[23] Huh YS, Jeon SJ, Lee EZ, Park HS, Hong WH, Korean J. Chem. Eng., 28(3), 633 (2011)

[24] Parolo C, Merkoci A, Chem. Soc. Rev., 42(2), 450 (2013)

[25] Gomez FA, Bioanalysis, 6(21), 2911 (2014)

[26] Martinez AW, Phillips ST, Whitesides GM, Carrilho E, Anal. Chem., 82(1), 3 (2010)

[27] Jokerst JC, Adkins JA, Bisha B, Mentele MM, Goodridge LD, Henry CS, Anal. Chem., 84(6), 2900 (2012)

[28] Carrilho E, Martinez AW, Whitesides GM, Anal. Chem., 81(16), 7091 (2009)

[29] Martinez AW, Phillips ST, Wiley BJ, Gupta M, Whitesides GM, Lab Chip, 8(12), 2146 (2008)

[30] Fujii I, Iwabuchi Y, Teshima T, Shiba T, Kikuchi M, Biorg. Med. Chem., 1(2), 147 (1993)

[31] Vella SJ, Beattie P, Cademartiri R, Laromaine A, Martinez AW, Phillips ST, Mirica KA, Whitesides GM, Anal. Chem., 84(6), 2883 (2012)

[32] Cheng CM, Martinez AW, Gong J, Mace CR, Phillips ST, Carrilho E, Mirica KA, Whitesides GM, Angew. Chem.-Int. Edit., 49(28), 4771 (2010)

[33] Saito M, Hagita H, Iwabuchi Y, Fujii I, Ikeda K, Ito M, Histochem. Cell Biol., 117(5), 453 (2002)

[34] Minami A, Otsubo T, Ieno D, Ikeda K, Kanazawa H, Shimizu K, Ohata K, Yokochi T, Horii Y, Fukumoto H, PLoS One, 9(1), e81941 (2014)

[35] Michaelis L, Menten ML, Biochem. z, 49(333-369), 352 (1913)

[36] Fanjul-Bolado P, Gonzalez-Garcia MB, Costa-Garcia A, Anal. Bioanal. Chem., 386(6), 1849 (2006)