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研究生:林佳蓁
研究生(外文):Chia-Chen Lin
論文名稱:應用奈米金球與奈米磁珠建立電化學免疫分析方法以檢測芒果主要過敏原–Man i 1
論文名稱(外文):Development of electrochemical immunoassay using gold nanoparticles and magnetic nanoparticles for the detection of a major mango allergen – Man i 1
指導教授:溫曉薇
口試委員:吳靖宙鄧秉華
口試日期:2021-07-19
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:107
中文關鍵詞:芒果過敏原Man i 1電化學免疫分析法奈米金球電化學免疫分析方法奈米磁珠
外文關鍵詞:Mango allergenMan i 1electrochemical immunosensorgold nanoparticlesimmunomagnetic nanoparticles
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摘要 i
Abstract ii
Content iii
Figure content vi
Table content viii
Abbreviations ix
Chapter I: Literature review 1
1.1 Mango allergies 1
1.1.1 Food allergy 1
1.1.2 Characteristics of mango allergy 2
1.1.3 Mango allergens 4
1.1.4 Assays for detecting food allergens 5
1.2 Electrochemical biosensor 11
1.2.1 Principle and approaches of electrochemistry 11
1.2.2 Electrochemical impedance spectroscopy (EIS) 13
1.2.3 Amperometry (i-t) 15
1.2.4 Screen-printed carbon electrode (SPCE) 17
1.3 Nanomaterials 22
1.3.1 Gold nanoparticles (AuNPs) 22
1.3.2 Immunomagnetic nanoparticles (IMNPs) 24
1.3.3 Applications of nanomaterials in electrochemical immunosensors 25
Chapter II: Materials and methods 29
2.1 The objective of the study 29
2.2 Materials and instruments 30
2.2.1 Materials 30
2.2.2 Instruments 31
2.3 Preparation of antigen 31
2.3.1 Sample extraction 31
2.3.2 Expression and purification of rMan i 1 31
2.3.3 Purification of antibody 32
2.4 Modification of SPCE 34
2.4.1 Preparation of IMNPs 34
2.4.2 Preparation of Antigen-IMNPs complexes 34
2.4.3 Fabrication of the immunosensor 35
2.4.4 Impedimetric measurement 37
2.4.5 Amperometric measurement 37
2.5 Assay optimization 38
2.6 Assay performance 38
2.6.1 Sensitivity and specificity 38
2.6.2 The influence of food matrix 39
2.6.3 Effect of heat treatment 39
2.7 Accuracy and precision of the developed assay 40
2.8 Real food sample analysis 40
2.9 Statistical Analysis 40
Chapter III: Results and discussion 42
3.1 Purification of recombinant mango allergen-rMan i 1 42
3.2 Purification of antibody 43
3.3 Optimization of assay conditions 44
3.3.1 Modification of SPCE 44
3.3.1.1 Optimization of electrodeposition time of AuNPs-modified SPCE 44
3.3.1.2 Optimization of the size of AuNPs 45
3.3.1.3 Optimization of the blocking buffer on the SPCE 45
3.3.1.4 Optimization of the concentration and incubation time of L-cysteine 45
3.3.2 Reactions with IMNPs 46
3.3.2.1 Comparison of three assay models 47
3.3.2.2 Optimization of blocking buffer of IMNPs 47
3.3.2.3 Optimization of antibody pairing on the SPCE and IMNPs 48
3.3.2.4 Optimization of antibody concentration on the SPCE 48
3.3.2.5 Optimization of the size and antibody concentration on the IMNPs 49
3.3.2.6 Optimization of the volume ratio of IMNPs and samples 50
3.3.2.7 Optimization of incubation time for IMNPs with samples 50
3.3.3 Usage of HRP 51
3.3.3.1 Optimization of the pH of HQ solution 51
3.3.3.2 Optimization of the different molar ratio of H2O2/HQ 52
3.3.3.3 Optimization of incubation time for Man i 1-IMNPs complexes on the SPCE 52
3.3.3.4 Optimization of concentration and incubation time of HRP labelled 52
3.4 Sensitivity of Man i 1 detection 53
3.5 Specificity of Man i 1 detection 54
3.6 Matrix effect in food samples 54
3.7 Determination of Man i 1 in the influence of heat treatment 55
3.8 Precision and accuracy of immunosensor (Inter/ Intra assay) 56
3.9 Identification of Man i 1 from food samples 56
Chapter IV: Conclusion and future work 58
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