||Development of an Inverse Opal Photonic Crystal Biosensing Device with Enhanced Fluorescence
||Department of BioMedical Engineering
近年來，糖尿病視網膜病變（Diabetic Retinopathy, DR）是視力喪失的主要原因。然而，由於繁瑣的檢查程序，目前該疾病的篩檢率仍然很低。因此，簡易評估和低成本的生醫感測裝置變得越來越重要。根據之前的文獻指出，淚脂質運載蛋白（Lipocalin 1, LCN1）是糖尿病視網膜病變的生物指標性蛋白之一，其對健康人的濃度水平約為1-2 mg / mL;而增殖型糖尿病視網膜病變（Proliferative Diabetic Retinopathy, PDR）患者中的LCN1濃度比健康的受試者高出幾倍。因此，用於DR的長期監測（8~10小時）的非侵入性和低成本生物感測裝置變得越來越重要。而光子晶體在近年來已被廣泛用於生物傳感器中。先前研究表明，光子晶體可用來測量材料中的折射率變化與增顯螢光的強度。為了滿足上述感測裝置的需求，本研究開發了一種具有螢光增顯的反蛋白石光子晶體生醫感測裝置。將一滴二氧化矽膠體懸浮液在PMMA模板上在室溫下風乾幾小時以進行自組裝。在懸浮液完全乾燥後，將聚（乙二醇）二丙烯酸酯（PEG-DA，Mn = 250）加入PC結構中並用UV光固化5分鐘。隨後，在用BOE蝕刻PC之後獲得反蛋白石PC結構。由於在反蛋白石基質內形成夾心的免疫複合物，使得感測裝置的折射率發生變化，並發生反射波長的偏移與螢光信號的增強。在初步結果中，在蛋白濃度0.1 mg / mL下的螢光強度比對照組高出1.5倍，而在樣品乾燥的條件下波長偏移量為7.1 nm。結果與先前的實驗結果一致。我們也使用反蛋白石生物感測器去測試不同濃度的LCN 1蛋白。結果顯示，在0.1 mg / mL的組別螢光強度增加大約14.5，在0.05 mg / mL的組別增加8.5，而在0.01 mg / mL的組別增加7。因此，該裝置檢測蛋白質濃度的極限為0.01 mg / mL。另外，在這項研究中，我們還開發了一種創新的生物感測器。該裝置在Pluronic F-127水凝膠當中嵌入螢光染料和捕獲抗體。當抗原溶液浸入至水凝膠層中時，螢光染料和捕獲抗體釋放到反蛋白石層。之後，免疫反應將在反蛋白石層中進行。結果顯示，在蛋白濃度0.15 mg / mL的組別螢光強度增加16，0.1 mg / mL的組別增加8，而0.05 mg / mL的組別則增加4。因此，該反蛋白石生醫感測器偵測蛋白濃度達到0.05 mg / mL。另一方面，我們也使用了帶有藍色濾光片的手機相機拍攝照片，並利用影像螢光強度檢測不同蛋白濃度的樣品。實驗數據顯示在濃度0.15 mg / mL組別的螢光強度高於0.1 mg / mL和0.05 mg / mL的組別。然而，只有0.15 mg / mL的組別才能識別其差異。這項研究提供了一個具有前瞻性的光學生物感測器，並且在不久的將來應用於糖尿病視網膜病變的篩選上。
Diabetic retinopathy (DR) is a major cause of vision loss in the modern time. Nevertheless, the current screening rate stays low because of the tedious examination procedure. In the previous study, lipocalin 1 (LCN 1), was found to be a key biomarker in the healthy human tear (approximately 1-2 mg/mL). By contrast, the concentration of LCN1 in the proliferative diabetic retinopathy (PDR) patients rises several folds higher than other healthy counterparts. As a result, a non-invasive and low-cost biosensing device for long-term monitoring (8~10 h) of DR becomes increasingly essential. Photonic crystal (PC) has been widely used in biosensors in the recent years. Prior research showed that PC could be used to measure refractive index changes in material and enhance fluorescence intensity. To meet the abovementioned demands in biosensors, an inverse-opal PC biosensing device with enhanced fluorescence was then developed in this study. We used silica particles (diameter: 300 nm) to fabricate an inverse-opal PC substrate. A drop of silica colloidal suspension was air dried on a PMMA template few hours at room temperature to enable self-assembly. After the suspension was completely dried out, poly(ethylene glycol) diacrylate (PEG-DA, Mn=250) was added into the PC structure and cured with UV light for 5 min. Subsequently, an inverse-opal PC structure was obtained after etching the PC with BOE. As the refractive index of the inverse opal PC changed because of the sandwiched immuno-complexes formed within the PC matrix, the reflected wavelength shifted and signal fluorescence was enhanced. In the preliminary result, the fluorescence intensity of 0.1 mg/mL was enhanced 1.5 times higher than the control while the wavelength shift was 7.1 nm in a dry condition. The result was consistent with the prior work. We also use inverse opal PC biosensor to test different concentration of LCN 1 protein. The results showed that fluorescence intensity increased in the 0.1 mg/mL group by 14.5, the 0.05 mg/mL group increased by 8.5, and the 0.01 mg/mL group increased by 7. Hence, the limit of detection for the protein concentration achieved with this device was 0.01 mg/mL.
In this research, we also develop an innovative biosensor. This device embedded fluorescent dye and captured antibody in a Pluronic F-127 hydrogel. When the antigen solution immersed into the hydrogel layer, the fluorescent dye and capture antibody released into the inverse opal layer. Afterward, the biosensor would conduct the immunoreaction in the inverse opal layer. The results showed that the fluorescence intensity increased in the 0.15 mg/mL group by 16, the 0.1 mg/mL group increased by 8, and the 0.05 mg/mL group increased by 4. Therefore, the limit of detection of this inverse opal PC biosensor achieved 0.05 mg/mL. On the other hand, we used a mobile phone camera with the blue filter to detect the different concentration of samples. The experimental data showed that the 0.15 mg/mL group's fluorescence intensity is higher than the 0.1 mg/mL and 0.05 mg/mL group. However, only the 0.15 mg/mL group could recognize its difference. Eventually, this thesis provides a promising optical biosensor for hassle-free DR screening in the near future.
LIST OF TABLES.....VII
LIST OF FIGURES.....VIII
CHAPTER 1 INTRODUCTION.....1
1.1 Motivation and Overview....1
1.2 Inverse Opal Photonic Crystal....2
1.2.1 Bragg’s Law.....2
1.2.2 Principle of Inverse Opal Photonic Crystal..3
1.2.3 Mechanism of the Inverse Opal and Inverse Opal
Embedded AuNPs Photonic Crystal Enhanced
1.3 Diabetic Retinopathy....12
1.4 Biomarker Diagnosis in Human Tear Fluid...13
1.5 Aims and Contribution of the Thesis...14
CHAPTER 2 MATERIALS AND METHODS...15
2.1 Experimental Setup....15
2.2 Chip Configuration....17
2.3 Chip Fabrication and Protocol...19
2.3.1 Protocol for Inverse Opal Chips Fabrication..21
2.3.2 Protocol for Modified Probe Antibody on Chip.22
2.3.3 Mechanism of Inverse Opal Sandwiched Immunoassay
2.3.4 Protocol for Inverse Opal Sandwiched Immunoassay
2.3.5 Protocol for Hydrogels with BSA Protein (BCA
2.3.6 Mechanism of Inverse Opal Sandwiched Immunoassay
in Pluronic F-127 Hydrogel...26
2.3.7 Protocol for Inverse Opal Sandwiched Immunoassay
in Pluronic F-127 Hydrogel...28
CHAPTER 3 RESULTS AND DISCUSSION....30
3.1 Inverse opal Chip.....30
3.1.1 Silica Particles Self-Assembly with Face-Centered
3.1.2 Spectrum of Inverse opal...31
3.1.3 Inverse Opal Embedded Different Size of AuNP.34
3.2 Effects of Inverse Opal Enhanced Fluorescence.35
3.2.1 Concentration of Dye 488 Enhancement...35
3.3 Effects of Protein Concentration Wavelength Shift.39
3.4 Inverse opal PC Biosensor for Sandwich Immunoassay..41
3.4.1 Preliminary Testing of Biosensor Function..41
3.4.2 Time Effect of Sandwich Immunoassay on Inverse
3.4.3 Detect Different Protein Concentration of
3.4.4 Detect Different Protein Concentration with
Inverse Opal Embedded AuNP Biochip...49
3.5 Effects of Protein Diffusion in Hydrogels...51
3.5.1 Pluronic F-127 and pHEMA Release Rate..51
3.5.2 Sandwich Immunoassay with Pluronic F-127 Hydrogel
3.5.3 Different Protein Concentration of Sandwich
Immunoassay with Pluronic F-127 Hydrogel.55
3.6 Detect Different the Fluorescence Intensity with mobile
CHAPTER 4 CONCLUSION....60
CHAPTER 5 FUTURE WORK.....62
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