Integrative bioanalysis using LSPR-driven dual sensing technologies for enhancing detection and precision

국문 초록 (Abstract)

본 논문은 고도화된 색 변화 기반 및 이중 모드 바이오센서 개발을 위한 핵심 메커니즘으로서 국소 표면 플라스몬 공명(Localized Surface Plasmon Resonance, LSPR)의 응용에 대한 심층적 연구를 제시한다. LSPR은 금속 나노구조와 빛의 상호작용에서 발생하는 현상으로, 분석 물질(analyte) 결합에 따른 가시적 색 변화 반응을 유도하는 다양한 광학 센서의 기반 원리로 활용된다. 본 연구는 나노입자의 조성, 형태, 그리고 주변 유전체 환경이 LSPR의 스펙트럼 특성과 감도에 미치는 영향을 중심으로 LSPR의 기초 이론을 포괄적으로 고찰하는 것으로 시작된다. 이러한 이론적 기반을 바탕으로, 본 논문은 LSPR을 색 변화(colorimetric) 및 형광(fluorometric) 센서 플랫폼에 통합하는 다양한 전략을 탐색하며, 특히 색 변화와 형광 신호를 동시에 활용하는 이중 모드 시스템에 주목한다. 이와 같은 이중 신호 플랫폼은 검출 정확도 향상, 위양성 감소, 자가보정 기능을 가능하게 하며, 실제 생체 진단에 적용되어 요소(urea) 및 티로시나아제(tyrosinase)와 같은 임상 관련 바이오마커를 효과적으로 검출하였다. 첫 번째 바이오센서는 고감도 형광-색 변화 하이브리드 탄소 점 기반 나노센서로, 요소를 이중 모드로 모니터링한다. 요소가 요소분해효소(urease)에 의해 분해되어 생성되는 이산화탄소와 암모니아는 용액의 pH를 증가시키며, 이는 탄닌산의 환원력을 활성화시켜 플라스몬 은 나노입자(AgNPs)를 in situ로 생성하게 한다. 생성된 AgNPs의 흡수 스펙트럼은 탄소 점(CDs)의 형광 스펙트럼과 중첩되어, 요소 존재 시 형광이 효과적으로 소광된다. 해당 시스템은 형광 기반으로는 18 nM, 색 변화 기반으로는 1.05 μM의 낮은 검출한계를 보였으며, 각각 100 nM–1 mM 및 50 µM–1 mM 범위에서 우수한 직선성을 나타내어 현재까지 보고된 센서 중 가장 낮은 검출한계를 기록하였다. 두 번째 바이오센서는 LSPR 파장 이동 기반의 개폐형 형광 신호를 이용하는 새로운 비율형 형광 분석법을 제시하며, 흑색종 진단에 활용되었다. 이 시스템은 다중 색 변화(multicolorimetric) 및 비율형 형광 검출(ratiometric fluorometric detection)을 통합한 초고감도 티로시나아제 정량 플랫폼이다. 금 나노바이피라미드(AuNBPs)의 조절 가능한 LSPR과 상향변환 나노입자(UCNPs)를 기반으로 한 FRET 기전을 활용하며, 티로시나아제에 의해 카테콜이 산화되어 퀴논을 형성하면서 Ag의 침착이 억제되어 농도에 따른 색 변화가 유도된다. 이 방식은 색 변화 및 형광 감지에서 각각 4.13 × 10⁻⁵ U/mL 및 2.71 × 10⁻⁵ U/mL의 우수한 검출한계를 보이며, 복잡한 생물학적 샘플에서도 높은 선택성과 안정성을 입증하였다. 마지막으로, 별도의 라벨링 및 희석 없이 원뇨(raw urine) 내 요소를 직접 검출할 수 있는 플라스몬-양자점 하이브리드 분석 시스템이 개발되었다. 이 시스템은 urease 효소에 의한 요소의 가수분해로 생성된 암모니아가 국소적인 pH 변화를 유도하며, 이는 카테콜의 환원 활성화 및 금 나노바이피라미드 표면에서의 은 침착 효율을 조절한다. 변화된 LSPR은 AuNBPs와 양자점(QDs) 간의 FRET를 조절하여 형광 채널 중 하나를 선택적으로 소광시킨다. 해당 이중 모드 플랫폼은 QDs 기반 비율형 형광과 LSPR 변조를 결합하여 복잡한 샘플 전처리 없이도 민감하고 정확한 요소 검출이 가능함을 보여준다. 이처럼, 본 논문에서 제시된 LSPR 기반 이중 감지 바이오센서는 고감도, 고선택성, 높은 실용성을 동시에 갖추고 있으며, 향후 현장 진단(point-of-care) 및 환경 모니터링 등의 분야에 폭넓게 응용될 수 있는 가능성을 보여준다.

다국어 초록 (Multilingual Abstract)

Abstract
This thesis presents an in-depth investigation into the application of Localized Surface Plasmon Resonance (LSPR) as a core mechanism for the development of advanced dual-mode biosensors. The work explores the dual-mode systems that combine LSPR-based color change with complementary fluorescence signals to enhance detection accuracy, reduce false positives, and enable self-calibrating responses. These dual-signal platforms were applied to real-world biomedical diagnostics, highlighting their promise for future utilization in point-of-care diagnostics and environmental monitoring. The first biosensor is a highly-sensitive fluorometric and colorimetric hybrid carbon-dot nanosensor for dual monitoring of urea. The research wherein urease enzyme can specifically hydrolyze urea to generate carbon dioxide and ammonia, causing an increase in the pH, which activates the reduction affinity of tannic acid to generate plasmonic AgNPs in situ. The absorption spectra of the AgNPs overlapped closely with the fluorescence spectrum of the CDs, enabling effective quenching of the CD fluorescence upon urea exposure. This fluorogenic and chromogenic dual-signal is used to quantify the accurate urea concentrations, showing limit-of-detection (LOD) of 18 nM and 1.05 μM for fluorometric and colorimetric sensing, respectively. The second biosensor introduces a novel ratiometric fluorometric assay, employing LSPR wavelength shift-driven on-off signals for melanoma diagnostics. This method leverages a dual-mode biosensing platform integrating multicolorimetric and ratiometric fluorometric detection for ultra-sensitive tyrosinase quantification. This platform leverages LSPR modulation of gold nanobipyramids (AuNBPs) alongside a fluorescence resonance energy transfer (FRET)-based mechanism using upconversion nanoparticles (UCNPs). The tunable LSPR of AuNBPs provided a bidirectional ratiometric multicolor emission improving sensitivity and selectivity. Tyrosinase-mediated oxidation of catechol to quinone inhibits silver deposition on AuNBPs, inducing a distinct concentration-dependent color shift for precise visual quantification. This dual-modality assay achieves an exceptional LOD of 4.13 × 10-5 and 2.71 × 10-5 U/mL for colorimetric and fluorometric sensors, respectively, demonstrating high selectivity, robustness in complex biological samples. Lastly, a plasmonic-quantum dot hybrid assay is introduced for label-free, dilution-free urea detection in raw urine. This system utilizes the catalytic properties of urease to hydrolyze urea into ammonia and carbon dioxide, creating a localized pH shift that induces the reduction ability of catechol indirectly governs silver deposition efficiency on the AuNBP surface. In undiluted urine, this reaction dynamically alters the LSPR behavior of AuNBPs, which modulates the FRET between the QDs and the AuNBPs. The proximity-induced energy transfer leads to selective quenching of one fluorescence emission channel, enabling highly sensitive urea detection without the need for dilution or complex sample preparation. This dual-mode platform, combining LSPR modulation and ratiometric fluorescence using QDs, exhibits high sensitivity and reliability for direct urea detection in undiluted urine. Ultimately, these innovative biosensors, leveraging LSPR principles, exhibit exceptional sensitivity, selectivity, and applicability across diverse fields, promising rapid and accurate analyte detection for various diagnostic and monitoring applications.

Conclusion
This dissertation presents a comprehensive exploration of localized surface plasmon resonance (LSPR)-driven dual-mode biosensing strategies for high-sensitivity bioanalysis. By integrating colorimetric and fluorometric signal transduction mechanisms, we developed multifunctional nanohybrid platforms tailored for real-time, label-free detection of clinically and environmentally significant analytes. The research journey begins by the first platform that features nitrogen-doped carbon dots (N-CDs) and plasmonic silver nanoparticles (AgNPs) for dual-signal urea detection, enabling ratiometric fluorescence quenching via FRET and LSPR-based colorimetric modulation. The system exhibited exceptional sensitivity (LOD: 18 nM fluorometric, 1.05 µM colorimetric) and selectivity in complex matrices, including human urine. The second system employs gold nanobipyramids (AuNBPs) and upconversion nanoparticles (UCNPs) for tyrosinase-based melanoma diagnostics. Tyrosinase-catalyzed catechol oxidation governs silver deposition on AuNBPs, modulating LSPR shifts and dual-peak UCNP fluorescence via inverse ratiometric switching. This sensor achieved a remarkably low detection limit (LOD: 4.13 × 10-5 U/mL) and demonstrated high precision in human serum. The third platform overcomes the chloride interference barrier in raw urine sensing by employing a stabilized [Ag(NH3)2]+ complex and crown ether ligands to facilitate silver shell growth on AuNBPs for dilution-free urea quantification. Combined with dual-emissive quantum dot nanohybrids, this system achieved robust multicolorimetric and ratiometric responses with minimal background interference. All sensing platforms were successfully immobilized into hydrogel matrices, enabling portable, point-of-care (POC) biosensing with excellent stability and reproducibility. Collectively, these LSPR-driven dual-mode biosensors address key challenges in sensitivity, matrix compatibility, and signal reliability, offering powerful solutions for clinical diagnostics, food safety, and environmental monitoring.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1. Introduction of sensor and plasmonic sensor 1
• 1.2. Thesis outline 6
• Chapter 2. Literature review 7
• 2.1. Current state-of-art 7
• 2.2. Surface Plasmon Resonance (SPR) 8
• 2.3. Localized Surface Plasmon Resonance (LSPR) 9
• 2.4. Colorimetric and Fluorometric LSPR Biosensors 10
• 2.5. Gold-Based Nanoplasmonic Biosensors 12
• 2.6. Silver-Based Nanoplasmonic Biosensors 19
• 2.7. Sensing Strategies Utilizing LSPR-Active Nanomaterials 21
• 2.7.1. Aggregation-Based LSPR Colorimetric Assays 23
• 2.7.2. Aggregation Suppression-Based LSPR Colorimetric Assays 25
• 2.7.3. Etching-Driven Colorimetric Sensing Strategies 26
• 2.7.4. Growth-Mediated Colorimetric Detection Strategy 28
• 2.8. Types and Mechanisms of Fluorescence Quenching 30
• 2.8.1. Vibrational Relaxation and Internal Conversion 32
• 2.8.2. Intersystem Crossing 33
• 2.8.3. Frster Resonance Energy Transfer (FRET) 35
• 2.8.4. Dexter Electron Transfer (DET) 36
• 2.8.5. Triplet-Triplet Annihilation (TTA) 37
• 2.8.6. Radiative Energy Transfer 38
• 2.9. Summary 40
• Chapter 3. Fluorometric and Colorimetric Hybrid Carbon-Dot Nanosensors for Dual Monitoring of Urea 42
• 3.1. Introduction 42
• 3.2. Experimental section 45
• 3.2.1. Reagents and chemicals 45
• 3.2.2. Characterizations 45
• 3.2.3. Synthesis and purification of Carbon dots 45
• 3.2.4. Urea Sensing 46
• 3.2.5. Hydrogel Immobilizations 46
• 3.2.6. Urine Sample Monitoring 47
• 3.3. Results and Discussion 47
• 3.3.1. Validation of urea assay biosensor 47
• 3.3.2. Optimization of urea biosensor 56
• 3.3.3. The biosensor response upon urea concentration variations 58
• 3.3.4. Selectivity test for urea assay 62
• 3.3.5. Versatility and reliability of the dual signaling CD hybrid nanosensor 63
• 3.3.6. Standard urea kit (standard quantitative colorimetric urea assay kit (DIUR-100)) 69
• 3.4. Chapter conclusion 74
• Chapter 4. Integrated Colorimetric and Fluorescent Assay Using Gold Nanobipyramids and UCNPs for Tyrosinase-Based Melanoma Diagnostics 75
• 4.1. Introduction 75
• 4.2. Materials and Methods 78
• 4.2.1. Materials 78
• 4.2.2. Instrumentation 79
• 4.2.3. Synthesis of gold nanobipyramids (AuNBPs) 79
• 4.2.4. Synthesis of core-shell upconversion nanoparticles coated Phospholipids (UCNPs@Lipo) 80
• 4.2.5. Tyrosinase Assay 82
• 4.2.6. Serum sample assay 83
• 4.2.7. Inhibitor screening 83
• 4.3. Results and Discussion 84
• 4.3.1. Tyrosinase sensor validation 84
• 4.3.2. Multicolorimetric assay of tyrosinase 89
• 4.3.3. Selectivity and interference investigation 98
• 4.3.4. Sensor validation in serum 100
• 4.3.5. Monitoring the inhibition efficiency of kojic acid 102
• 4.4. Chapter conclusion 105
• Chapter 5. PlasmonicQuantum Dot Hybrid Assay for Label-Free, Dilution-Free Urea Detection in Raw Urine Samples 107
• 5.1. Introduction 107
• 5.2. Experimental section 111
• 5.2.1. Materials 111
• 5.2.2. Synthesis of ZnCdS QDs 111
• 5.2.3. Synthesis of Cu-doped ZnCdS QDs 112
• 5.2.4. Synthesis of Red Fluorescent QD-Doped Silica Nanoparticles 112
• 5.2.5. Synthesis of Dual-Emitting Red QDs@Silica@Green QDs Nanohybrid 113
• 5.2.6. Synthesis of Gold Nanobipyramids (AuNBPs) 113
• 5.2.7. Direct and Dilution-free Urea Assay 114
• 5.2.8. Hydrogel Matrix for Point-of-Care (POC) Usability 115
• 5.3. Result and discussion 116
• 5.3.1. Optical Characteristics of AuNBPs and QD Nanohybrids 116
• 5.3.2. Dual-Mode Urea Detection: Colorimetric and Fluorometric Response 121
• 5.3.3. Analytical Performance Evaluation 127
• 5.3.4. Point-of-Care Applicability 128
• 5.4. Chapter conclusion 130
• Chapter 6. Dissertation Conclusion 131
• 6.1. Conclusion 131
• 6.2. Future directions 132
• 6.2.1. Plasmonic-enhanced fluorescence (PEF) 132
• 6.2.2. Dual-Mode Plasmon-Enhanced Fluorescence Sensors Based on Distance and Plasmon Wavelength Dependence Using Shell-Isolated Nanoparticle-Enhanced Fluorescence (SHINEF) 134
• References 138