표면증강라만 래피드 키트를 이용한 인플루엔자 바이러스 A의 조기진단

Abstract

본 연구에서는 지난 2009년 세계적으로 대유행 하였던 인플루엔자 바이러스 A/H1N1pdm를 저농도에서 빠른 시간 내 검출할 수 있는 표면증강라만 산란(surface-enhanced Raman scattering (SERS)) 기반의 래피드 키트를 개발하였다. 인플루엔자 바이러스는 급성 호흡기 질환을 일으키는 전염성이 강한 바이러스로, 백신 치료와 대유행을 막기 위해 빠르게 조기진단하는 것이 중요하다. 현재 인플루엔자 바이러스 확진 검사 (세포배양, real-time RT-PCR, ELISA)는 감염 결과를 확인하기까지 하루 이상이 소요된다. 반면, 인플루엔자 신속 진단 검사 (rapid influenza diagnostic test, RIDT)는 진료현장에서 30분 이내 인플루엔자 감염을 진단할 수 있지만, 민감도가 낮아 이 결과만으로 확진 하기 어렵다. 따라서, 빠르고 간단하며 조기진단이 가능한 검사법의 개발이 지속적으로 요구되고 있으며, 이러한 문제점들을 해결하기 위해 고감도 분석이 가능한 SERS 기술을 래피드 키트에 적용하였다. 인플루엔자 바이러스 A를 검출할 수 있는 항체와 SERS 신호를 내는 라만 신호 분자를 금 나노 입자에 흡착하여, 기존의 상용화 래피드 키트에서 사용되는 금 나노 입자를 대체하였다. 본 SERS 기반의 래피드 키트를 이용하면 인플루엔자 바이러스 A를 금 나노 입자 응집에 의한 색 변화에 의해 눈으로 검출할 수 있을 뿐만 아니라 SERS 신호측정을 통해 낮은 농도까지 검출할 수 있으며, 정확한 정량분석 또한 가능하였다. 이 키트를 이용하여 인플루엔자 바이러스 A를 눈으로는 1 x 10^3 PFU/mL까지, SERS 신호 측정을 통해 304.65 PFU/mL까지 측정가능 하였다. |In this study, we developed a surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay kit for rapid detection of an influenza virus A/H1N1pdm at early stage. Influenza virus is a highly infectious virus that causes acute respiratory diseases. Thus, it is important to early diagnose the virus to prevent a pandemic as well as to cure the infected patient with an adequate vaccine. Current diagnostic tests (virus cell culture, real-time RT-PCR, and ELISA) take longer than one day to confirm the disease. In contrast, rapid influenza diagnostic test (RIDT) is possible to detect the infection within 30 minutes, but it is hard to confirm the viral infection only with this test because of its low sensitivity. Therefore, the development of rapid, simple test method for early diagnosis of flu virus is urgently needed to solve these problems. For these reasons, the SERS technique, which is possible to detect analytes with high sensitivity, can be applied to lateral flow immunoassay kit. The gold nanoparticles (GNPs) used in commercial rapid kit are replaced with SERS-active GNPs that are manufactured by adsorption of Raman reporter and anti-influenza A antibodies on the GNPs. Using these novel nanoprobes, it is possible to detect the influenza virus A not only by naked eyes but also by SERS measurement. The color change of the test line caused by aggregation of GNPs is visually detectable over 1 x 10^3 PFU/mL. Additionally, the influenza virus A could be identified as lower as 304.65 PFU/mL by SERS measurement.; In this study, we developed a surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay kit for rapid detection of an influenza virus A/H1N1pdm at early stage. Influenza virus is a highly infectious virus that causes acute respiratory diseases. Thus, it is important to early diagnose the virus to prevent a pandemic as well as to cure the infected patient with an adequate vaccine. Current diagnostic tests (virus cell culture, real-time RT-PCR, and ELISA) take longer than one day to confirm the disease. In contrast, rapid influenza diagnostic test (RIDT) is possible to detect the infection within 30 minutes, but it is hard to confirm the viral infection only with this test because of its low sensitivity. Therefore, the development of rapid, simple test method for early diagnosis of flu virus is urgently needed to solve these problems. For these reasons, the SERS technique, which is possible to detect analytes with high sensitivity, can be applied to lateral flow immunoassay kit. The gold nanoparticles (GNPs) used in commercial rapid kit are replaced with SERS-active GNPs that are manufactured by adsorption of Raman reporter and anti-influenza A antibodies on the GNPs. Using these novel nanoprobes, it is possible to detect the influenza virus A not only by naked eyes but also by SERS measurement. The color change of the test line caused by aggregation of GNPs is visually detectable over 1 x 10^3 PFU/mL. Additionally, the influenza virus A could be identified as lower as 304.65 PFU/mL by SERS measurement.