생화학물질 다중센싱을 위한 표면-증강 라만 산란에 기초한 나노복합체 개발

Abstract

표면-증강 라만 산란 효과는 단분자 수준의 물질까지 검출이 가능한 고감도 측정 기술로 최근 많은 각광을 받고 있다. 표면-증강 라만 산란에 기초한 검출 기술은 검출 감도가 뛰어나고 여러 개의 시료를 동시에 검출 할 수 있는 장점으로 차세대 분석법으로 많은 연구가 되고 있다. 표면-증강 라만 산란 현상은 금속 표면의 전기장 공명과 전하의 이동 효과에 의해 나타나며, 1014-1015 배 가량 신호증강효과가 있다고 보고되어 왔다. 또한 표면 전자기장이 중첩되는 핫스팟이라는 현상을 이용하여 더욱 민감한 검출 기반을 마련 할 수 있다. 본 연구에서는 화학물질과 생물물질을 검출 할 수 있는 두 가지의 검출 툴을 구축하고자 하였다. 1 장에서는 이중 신호 유무기 나노복합체를 이용하여그램 음성 박테리아군인 E. coli J5와 F. tularensis에 대하여 표면-증강 라만 산란과 형광 기반의 다중검출을 수행하였다. 은 나노입자를 합성하고 핫스팟을 이용하기 위하여 라만 염료를 통해 응집체를 만들어 주었다. 응집된 나노입자는 전기수력학적 분사방법을 통하여 고분자 나노입자 내에 함입시켜 주었으며, 이 때 형광 염료를 첨가하여 주어 표면 증강 라만 산란-형광 이중 신호 나노복합체를 만들어 주었다. 표면-증강 라만 산란을 통하여 각 102 cells/mL의 농도까지 정량검출이 가능하였으며, 공초점 형광현미경을 통하여 빠르게 이미지를 확인 할 수 있었다. 또한 전기수력학적 분사방법을 통하여 제조한 자성 입자를 통하여 두 항원을 빠르게 검출 할 수 있었다. 2 장에서는 이방성 모양 나노입자와 자극 반응성을 가진 고분자 복합체를 이용하여 화학 물질의 검출이 가능한 표면-증강 라만 산란에 기초한 검출 기판을 제작하였다. 열적 자극반응성을 가진 N-isopropylacrylamide (NIPAM) 고분자와 이방성 모양의 금 나노 입자를 합성하여 양전하를 띄고 있는 poly(NIPAM-allylamonium chloride) (poly(NIPAM-AA))과 음전하를 띄고 있는 가지가 있는 형태의 금 나노 입자를 이용하여 유무기 복합체를 형성하였다. 금 나노입자 사이의 거리를 온도 자극 반응성을 가진 NIPAM을 이용하여 조절함으로써 핫스팟을 형성할 수 있었다. 그 결과 발암 물질인 malachite green을 0.012 ppm까지 검출이 가능하였으며, 금 나노 입자의 제조 시 포함되어 있는 RBITC 라만 염료를 기준 시료로 사용함으로써 정량과 정성이 한번에 가능한 화학 검출 기판을 제조 할 수 있었다. 요약하면, 본 연구에서는 표면 증강 라만 산란-형광 이중 신호를 가지는 유무기 나노복합체를 통하여 생물분자 및 병원균을 고감도로 다중 검출 할 수 있었다. 또한 이방성 모양의 나노입자와 열적 자극반응성을 가진 p(NIPAM-AA)를 이용하여 표면 증강 라만 신호를 극대화 하였으며, 이를 통하여 발암 물질인 malachite green을 고감도로 검출 하였다. 결론적으로, 본 연구를 통해서 광학에 기반한 현장에서 진단이 가능한 고감도 진단과 바이오센싱 및 바이오이미징 분야에 적용 가능한 유무기 복합체를 개발하였으며, 다양한 환경에서 안정적이고, 고감도로 생체 분자 및 화학 분자를 검출 할 수 있는 기술을 개발함으로써 잠재적인 화생방전 및 과학 수사에 필요한 현장에서 사용할 수 있는 바이오 및 화학 센서 개발에 크게 기여할 것으로 기대된다. |Surface enhanced Raman scattering (SERS) has been of great interest as a powerful tool to study vibrational information of molecules adsorbed on metal surfaces. SERS-based biological and chemical detection tool has been attracted in the field of the next generation of detection technique due to their high sensitivity and simultaneous detection of several pathogens. SERS phenomenon results from an electromagnetic effect and charge-transfer effect on metal surface and its enhancement factor is as much as 1014-1015. Moreover, hot spots are created due to overlap of electromagnetic effect. Thus, highly sensitive and multiplexed detection tools need to be developed. In chapter 1, we first report that SERS-FL based dual nanoprobes in the form of hierarchical nanostructures with metallic nanoparticle clusters (MNPCs) were prepared and used along with magnetic beads (MBs) for fast and multiplexed detection of bacterial pathogens. Both MNPCs with different Raman dyes and two sets of FL dyes were simultaneously encapsulated within polymeric nanoparticles using electrohydrodynamic (EHD) jetting and chemically stabilized. Two different sets of monoclonal antibodies (mAb) against two kinds of bacterial pathogens were separately conjugated with the dual nanoprobes and the MBs, respectively. Sandwich-type immunocomplexes composed of SERS-FL dual nanoprobes, pathogens, and MBs were formed in the presence of Escherichia coli (E. coli) J5 and Francisella tularensis (F. tularensis), representing a linear correlation between Raman intensity and pathogen concentration, and limit of detection of 102 cells/ml. Furthermore, selective sandwich-type immunocomplexes against pathogens were also imaged by FL signals at 514 nm and 633 nm wavelength for excitation. Conclusively, excellent capability of fast and multiplexed detection of bacterial pathogens was achieved using a new class of SERS-FL dual nanoprobes, providing a powerful tool for qualitative and quantitative multiplexed biodetection of several pathogens based on SERS and FL. In chapter 2, we have developed stimuli-responsive surface enhanced Raman scattering (SERS) substrates by using branched gold nanoparticles (bGNPs) and thermally-responsive poly(N-isopropylacrylamide-co-allylamonium chloride), poly(NIPAM-co-AA) for rapid and high sensitive chemical detection with highly reproducible results. These NPs having negative charges had ionic interaction with positively charged poly(NIPAM-co-AA) tethered on the patterned gold substrates. Thermally-triggered aggregation of poly(NIPAM-co-AA) resulted in increased SERS signal, potentially due to closer proximity between the bGNPs bound to the polymer chain. Furthermore, archetypal Raman peaks of rhodamine B isothiocyanate (RBITC) of the bGNPs were used as internal standards, and concentration of malachite green as a carcinogen was determined by comparing the relative intensity of carcinogen peaks with RBITC. Chemical concentration was quantitatively measured with high sensitivity by comparing Raman intensity of a chemical with that of RBITC. Therefore, these SERS substrates with thermally switchable electromagnetic coupling could be useful for highly sensitive chemical detection. In this thesis, we studied organic-inorganic nanocoposites applicable to fast diagnosis, bio-sensing and bio-imaging. We developed technologies for detection of biomolecules and chemicals with high sensitivity and stability in various environments. Highly sensitive multiplexed detection of biomolecules and pathogens was achieved via organic-inorganic nanocoposites with SERS-FL dual signals. In addition, SERS signal to detect malachite green was maximized by using branched gold nanoparticles along with thermal responsive poly(NIPAM-AA) tethered on the patterned gold substrates. In conclusion, these SERS-based nanocomposites have great potential for multiplexed biological and chemical detection.; Surface enhanced Raman scattering (SERS) has been of great interest as a powerful tool to study vibrational information of molecules adsorbed on metal surfaces. SERS-based biological and chemical detection tool has been attracted in the field of the next generation of detection technique due to their high sensitivity and simultaneous detection of several pathogens. SERS phenomenon results from an electromagnetic effect and charge-transfer effect on metal surface and its enhancement factor is as much as 1014-1015. Moreover, hot spots are created due to overlap of electromagnetic effect. Thus, highly sensitive and multiplexed detection tools need to be developed. In chapter 1, we first report that SERS-FL based dual nanoprobes in the form of hierarchical nanostructures with metallic nanoparticle clusters (MNPCs) were prepared and used along with magnetic beads (MBs) for fast and multiplexed detection of bacterial pathogens. Both MNPCs with different Raman dyes and two sets of FL dyes were simultaneously encapsulated within polymeric nanoparticles using electrohydrodynamic (EHD) jetting and chemically stabilized. Two different sets of monoclonal antibodies (mAb) against two kinds of bacterial pathogens were separately conjugated with the dual nanoprobes and the MBs, respectively. Sandwich-type immunocomplexes composed of SERS-FL dual nanoprobes, pathogens, and MBs were formed in the presence of Escherichia coli (E. coli) J5 and Francisella tularensis (F. tularensis), representing a linear correlation between Raman intensity and pathogen concentration, and limit of detection of 102 cells/ml. Furthermore, selective sandwich-type immunocomplexes against pathogens were also imaged by FL signals at 514 nm and 633 nm wavelength for excitation. Conclusively, excellent capability of fast and multiplexed detection of bacterial pathogens was achieved using a new class of SERS-FL dual nanoprobes, providing a powerful tool for qualitative and quantitative multiplexed biodetection of several pathogens based on SERS and FL. In chapter 2, we have developed stimuli-responsive surface enhanced Raman scattering (SERS) substrates by using branched gold nanoparticles (bGNPs) and thermally-responsive poly(N-isopropylacrylamide-co-allylamonium chloride), poly(NIPAM-co-AA) for rapid and high sensitive chemical detection with highly reproducible results. These NPs having negative charges had ionic interaction with positively charged poly(NIPAM-co-AA) tethered on the patterned gold substrates. Thermally-triggered aggregation of poly(NIPAM-co-AA) resulted in increased SERS signal, potentially due to closer proximity between the bGNPs bound to the polymer chain. Furthermore, archetypal Raman peaks of rhodamine B isothiocyanate (RBITC) of the bGNPs were used as internal standards, and concentration of malachite green as a carcinogen was determined by comparing the relative intensity of carcinogen peaks with RBITC. Chemical concentration was quantitatively measured with high sensitivity by comparing Raman intensity of a chemical with that of RBITC. Therefore, these SERS substrates with thermally switchable electromagnetic coupling could be useful for highly sensitive chemical detection. In this thesis, we studied organic-inorganic nanocoposites applicable to fast diagnosis, bio-sensing and bio-imaging. We developed technologies for detection of biomolecules and chemicals with high sensitivity and stability in various environments. Highly sensitive multiplexed detection of biomolecules and pathogens was achieved via organic-inorganic nanocoposites with SERS-FL dual signals. In addition, SERS signal to detect malachite green was maximized by using branched gold nanoparticles along with thermal responsive poly(NIPAM-AA) tethered on the patterned gold substrates. In conclusion, these SERS-based nanocomposites have great potential for multiplexed biological and chemical detection.