표면증강라만측정용 금 패턴 마이크로어레이 센서의 제작 및 응용

Abstract

최근 나노기술을 이용한 고감도 생체물질 검출법에 대한 관심이 높아지고 있다. 본 연구에서는 민감한 검출을 요구하는 바이오물질 즉 생체 표지자나 병원성 박테리아 검출을 위한 고감도 마이크로어레이 플랫폼 기술을 개발하였다. 기능성 금 할로우 나노입자를 합성하였고, 마이크로 어레이칩을 제작한 후 표면증강라만측정 기술을 통하여 바이오물질을 고감도로 정량 검출 하는 기술을 개발하였다. 기존 웰플레이트 기반의 효소결합면역흡착검사법(ELISA: Enzyme-linked Immuno Solvent Assay)은 검출한계가 높고, 시약소모가 많으며 고속 분석이 어렵다는 단점을 가지고 있다. 이러한 문제점을 해결하기 위해서 유리 기판 위에 항체나 항원을 어레이 형태로 고정하여 분석하는 마이크로 어레이 기반의 면역분석 기법이 오랜기간동안 연구 되고 있다. 본 연구에서는 이러한 마이크로어레이 기반의 면역분석법을 이용하여 바이오 물질에 대한 검출능력을 향상 시키고 효율적인 정량분석이 가능한 새로운 가치의 기술을 개발하는 것을 목표로 삼았다. 첫 장에는 기존의 면역분석법이 갖는 바이오마커 측정한계를 극복하기 위하여 표면증강라만측정을 기반으로 하는 면역분석 기술을 개발하였다. 서로 다른 자기조립박막을 도입한 친수성과 소수성을 갖는 마이크로어레이를 제작 하였다. 이 후에 금 할로우 나노입자를 합성하여 표면에 라만 표지자를 고정화한 다음 항원과 특이적으로 반응하는 항체를 고정화하여 기능성 나노 입자를 합성 하였다. 마이크로어레이의 친수성 패턴 표면에 항체를 고정화 한 후, 항원 적용하고 합성된 기능성 나노입자를 반응시키면 특이적인 면역반응을 통하여 검출물질이 센서표면에 고정화 된다. 이를 표면증강라만분광법으로 검출 함으로써 기존 분석기술과 비교하여 약 천 배이상으로 측정감도를 향상 하였고, 복잡한 실험과정의 간소화를 통해 시간을 단축하여 효과적으로 정량 검출을 수행 하였다. 두 번째 장에서는 첫 장에서 개발한 금 패턴 마이크로어레이 기술을 이용하여 또 다른 바이오 물질인 병원성 박테리아의 정량 검출을 수행하였다. 기존의 박테리아 검출법은 배양 과정을 반드시 거쳐야 하는 한계를 가지고 있다. 빠르고 민감한 방식의 박테리아 검출 기술을 위하여 하이브리드 마이크로 어레이칩 위에 박테리아를 고정화 한 후, 표면증강라만산란 분광법을 이용하여 병원성 박테리아를 효과적으로 정량 검출 하였다. 이러한 마이크로어레이 플랫폼 기반의 면역 분석 기술은 고감도 바이오마커, 병원성 박테리아 검출 진단에 유용하게 사용 될 수 있으며 식품 또는 생물학적 무기 검출 분석 분야에도 효율적으로 적용 될 수 있을 것으로 기대된다. |The interest in a fast and sensitive detection method for biological substances is increasing. In this study we assert that the application of vibrational spectroscopy is feasible for the detection of biological targets in the microarray device. We explored the use of surface-enhanced Raman scattering (SERS)-based microarray platform as immunoassay template for detection of biological substances, biomarker and bacteria. The enzyme-linked immunosorbent assay (ELISA) has been widely used as a standard immunoassay technique to detect the presence of biomarker and bacteria or its complementary antibody. However, there are significant limitations of conventional fluorescent dyes for their use in the immunoassay. First, typical fluorescence dyes used in immunoassay are susceptible to photobleaching and lack of sensitivity. Second, typical 96-well ELISA plate relatively large sample volume at least 100 μL is required. To overcome this limitation, surface-enhanced Raman scattering (SERS)-based microarray platform has been introduced as an alternative platform to explore protein biomarkers and pathogenic bacteria. We fabricated a hydrophobic/hydrophilic hybrid-patterned array that includes self-assemble monolayers (SAMs) for a rapid and sensitive immunoassay. Then, we developed a SERS-based immunoassay using a gold-patterned microarray chip for a highly sensitive and reliable detection of biological substance, human IgG biomarker and Streptococcus pneumonia. The limit of detection and working range for detectable concentration was significantly improved compared with the conventional method. The first chapter, we present a highly sensitive and rapid SERS-based immunoassay platform for the detection of human IgG biomarkers. For this purpose, a gold-patterned microarray chip has been fabricated and used as a SERS detection template. Two different types of self-assemble monolayers (SAM) were applied in the fabrication of hydrophilic well and hydrophobic background. Here, a typical sandwich immunocomplex protocol was adopted. Monoclonal antibodies were immobilized on gold patterned substrates, and then antigen solutions and polyclonal antibody-conjugated hollow gold nanospheres (HGNs) were serially added for the formation of sandwich immunocomplexes. Antigen biomarkers can be quantitatively assayed by monitoring the intensity change of a characteristic SERS peak of a reporter molecule adsorbed on the surfaces of HGNs. Under optimized assay conditions, the limits of detection (LOD) were determined to be 10 fg/mL. This detection technique can be applied in a wider dynamic concentration range with a better sensitivity compare to ELISA. This method, moreover, designed to simplify the laboratory procedures including spotting, washing, mixing and incubating steps. Therefore, all these advantages provided the current needs of consistency, reliability and sensitivity on the experiment result. Bacteria, especially of the Streptococcus pneumonia, cause a sepsis that the survival rate considerably decreases with every hour. In sepsis diagnostics, a fast and reliable identification of bacteria is crucial for safe lives or, at least to avoid severe complication. The bacteria detection by conventional method still requires too much time, and it is often detected too late because the cultivation step which needs several days. In the second chapter, we demonstrate the great potential of our approach by applying the highly sensitive detection of bacteria in the microarray device without cultivation process. For this purpose, we fabricated Raman-reporter embedded hollow gold nanospheres (HGNs) conjugated to an anti-Streptococcus pneumonia polyclonal antibody to enable a highly specific and sensitive SERS nanoprobe for the targeted detection of Streptococcus pneumonia. Monoclonal capturing antibodies were immobilized onto the microarray for targeting specific bacteria. Using these functionalized HGNs, the formation of sandwich immunocomplexes on the surface of gold-patterned wells was achieved. Target bacteria can be quantitatively monitored by the intensity change of characteristic SERS peaks of Raman reporters adsorbed on the surface of HGNs. Here, a gold-patterned microarray platform allows the detection of Streptococcus pneumonia at concentrations 1 CFU/mL without sample enrichment. This SERS-based immunoassay technique fulfills the current needs of high sensitivity detection method for bacteria which are essential for the clinical diagnosis of a disease.; The interest in a fast and sensitive detection method for biological substances is increasing. In this study we assert that the application of vibrational spectroscopy is feasible for the detection of biological targets in the microarray device. We explored the use of surface-enhanced Raman scattering (SERS)-based microarray platform as immunoassay template for detection of biological substances, biomarker and bacteria. The enzyme-linked immunosorbent assay (ELISA) has been widely used as a standard immunoassay technique to detect the presence of biomarker and bacteria or its complementary antibody. However, there are significant limitations of conventional fluorescent dyes for their use in the immunoassay. First, typical fluorescence dyes used in immunoassay are susceptible to photobleaching and lack of sensitivity. Second, typical 96-well ELISA plate relatively large sample volume at least 100 μL is required. To overcome this limitation, surface-enhanced Raman scattering (SERS)-based microarray platform has been introduced as an alternative platform to explore protein biomarkers and pathogenic bacteria. We fabricated a hydrophobic/hydrophilic hybrid-patterned array that includes self-assemble monolayers (SAMs) for a rapid and sensitive immunoassay. Then, we developed a SERS-based immunoassay using a gold-patterned microarray chip for a highly sensitive and reliable detection of biological substance, human IgG biomarker and Streptococcus pneumonia. The limit of detection and working range for detectable concentration was significantly improved compared with the conventional method. The first chapter, we present a highly sensitive and rapid SERS-based immunoassay platform for the detection of human IgG biomarkers. For this purpose, a gold-patterned microarray chip has been fabricated and used as a SERS detection template. Two different types of self-assemble monolayers (SAM) were applied in the fabrication of hydrophilic well and hydrophobic background. Here, a typical sandwich immunocomplex protocol was adopted. Monoclonal antibodies were immobilized on gold patterned substrates, and then antigen solutions and polyclonal antibody-conjugated hollow gold nanospheres (HGNs) were serially added for the formation of sandwich immunocomplexes. Antigen biomarkers can be quantitatively assayed by monitoring the intensity change of a characteristic SERS peak of a reporter molecule adsorbed on the surfaces of HGNs. Under optimized assay conditions, the limits of detection (LOD) were determined to be 10 fg/mL. This detection technique can be applied in a wider dynamic concentration range with a better sensitivity compare to ELISA. This method, moreover, designed to simplify the laboratory procedures including spotting, washing, mixing and incubating steps. Therefore, all these advantages provided the current needs of consistency, reliability and sensitivity on the experiment result. Bacteria, especially of the Streptococcus pneumonia, cause a sepsis that the survival rate considerably decreases with every hour. In sepsis diagnostics, a fast and reliable identification of bacteria is crucial for safe lives or, at least to avoid severe complication. The bacteria detection by conventional method still requires too much time, and it is often detected too late because the cultivation step which needs several days. In the second chapter, we demonstrate the great potential of our approach by applying the highly sensitive detection of bacteria in the microarray device without cultivation process. For this purpose, we fabricated Raman-reporter embedded hollow gold nanospheres (HGNs) conjugated to an anti-Streptococcus pneumonia polyclonal antibody to enable a highly specific and sensitive SERS nanoprobe for the targeted detection of Streptococcus pneumonia. Monoclonal capturing antibodies were immobilized onto the microarray for targeting specific bacteria. Using these functionalized HGNs, the formation of sandwich immunocomplexes on the surface of gold-patterned wells was achieved. Target bacteria can be quantitatively monitored by the intensity change of characteristic SERS peaks of Raman reporters adsorbed on the surface of HGNs. Here, a gold-patterned microarray platform allows the detection of Streptococcus pneumonia at concentrations 1 CFU/mL without sample enrichment. This SERS-based immunoassay technique fulfills the current needs of high sensitivity detection method for bacteria which are essential for the clinical diagnosis of a disease.