Untitled BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC BÁCH KHOA HÀ NỘI NGUYỄN THỊ NGUYỆT NGHIÊN CỨU TỔNG HỢP VẬT LIỆU CeO2 CÓ CẤU TRÚC NANO NHẰM ỨNG DỤNG TRONG CẢM BIẾN ADN LUẬN ÁN TIẾN SĨ KHOA HỌC VẬT LIỆU[.]
BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC BÁCH KHOA HÀ NỘI NGUYỄN THỊ NGUYỆT NGHIÊN CỨU TỔNG HỢP VẬT LIỆU CeO2 CÓ CẤU TRÚC NANO NHẰM ỨNG DỤNG TRONG CẢM BIẾN ADN LUẬN ÁN TIẾN SĨ KHOA HỌC VẬT LIỆU Hà Nội - 2020 BỘ GIÁO DỤC VÀ ĐÀO TẠO TRƯỜNG ĐẠI HỌC BÁCH KHOA HÀ NỘI NGUYỄN THỊ NGUYỆT NGHIÊN CỨU TỔNG HỢP VẬT LIỆU CeO2 CÓ CẤU TRÚC NANO NHẰM ỨNG DỤNG TRONG CẢM BIẾN ADN Ngành: Khoa học vật liệu Mã số: 9440122 LUẬN ÁN TIẾN SĨ KHOA HỌC VẬT LIỆU NGƯỜI HƯỚNG DẪN KHOA HỌC: PGS.TS PHƯƠNG ĐÌNH TÂM GS.TS TRẦN TRUNG Hà Nội - 2020 LỜI CẢM ƠN Trải qua năm học tập nghiên cứu hết mình, tác giả hoàn thành luận án Tiến sĩ Khoa học Vật liệu Viện AIST, trường Đại học Bách Khoa Hà Nội Để hồn thành luận án này, tác giả nhận hướng dẫn tận tâm, động viên bảo hết lòng hai thầy hướng dẫn PGS TS Phương Đình Tâm GS TS Trần Trung Qua đây, tác giả xin bày tỏ lòng biết ơn sâu sắc tới hai thầy đặc biệt PGS TS Phương Đình Tâm – người ln bên cạnh, sát sao, bảo, góp ý bước thực luận án tác giả Bên cạnh đó, tác giả nhận tư vấn, giúp đỡ, động viên vô to lớn thầy PGS TS Phạm Hùng Vượng, TS Nguyễn Đức Dũng PGS TS Đặng Thị Thanh Lê tồn thể thầy cô, bạn nghiên cứu sinh học viên cao học theo học Viện AIST anh chị em, bạn bè đồng nghiệp Tác giả xin gửi lời cảm ơn sâu sắc tới tồn thể thầy anh chị em bạn! Trong thời gian theo học Viện AIST, trường Đại học Bách Khoa Hà Nội, tác giả nhận tận tình giúp đỡ tập thể lãnh đạo Viện AIST, phòng ban chức trường Đại học Bách Khoa Hà Nội Tác giả xin trân trọng cảm ơn tất giúp đỡ này! Ngoài ra, tác giả xin tỏ lòng biết ơn sâu sắc tới đại gia đình nội ngoại hai bên, cảm ơn chồng tác giả đồng hành, tiếp sức cho tác giả suốt thời gian thực luận án! Tác giả Nguyễn Thị Nguyệt i LỜI CAM ĐOAN Tơi xin cam đoan cơng trình nghiên cứu riêng Các kết nêu luận án trung thực chưa tác giả khác công bố Hà Nội, ngày TM Tập thể hướng dẫn tháng năm 2020 Tác giả PGS.TS Phương Đình Tâm Nguyễn Thị Nguyệt ii MỤC LỤC LỜI CẢM ƠN i LỜI CAM ĐOAN ii MỤC LỤC iii DANH MỤC CÁC KÝ HIỆU VÀ CHỮ VIẾT TẮT vi DANH MỤC BẢNG ix DANH MỤC HÌNH x MỞ ĐẦU Chương 1: CẢM BIẾN ADN ĐIỆN HÓA 1.1 Giới thiệu chuỗi ADN 1.2 Cơ sở lý thuyết phương pháp điện hóa sử dụng nghiên cứu cảm biến ADN 1.2.1 Phương pháp quét vòng 1.2.2 Phương pháp quét xung vi phân (DPV) 11 1.2.3 Phương pháp qt sóng vng (SWV) 12 1.2.4 Phương pháp phổ tổng trở EIS 13 1.3 Các phương pháp cố định chuỗi ssADN 15 1.3.1 Hấp phụ vật lý 15 1.3.2 Cố định liên kết cộng hóa trị 16 1.3.3 Cố định thông qua tương tác Avidin/Streptavidin -Biotin 18 1.3.4 Cố định phương pháp điện hóa 19 1.4 Phương pháp phát lai hóa ADN 21 1.4.1 Phương pháp đánh dấu 21 1.4.2 Phương pháp không đánh dấu 25 1.5 Ứng dụng cảm biến sinh học 29 Kết luận chương 29 Chương 2: NGHIÊN CỨU TỔNG HỢP VẬT LIỆU THANH NANO CeO2 & NANO COMPOSIT CeO2@Ppy 31 2.1 Đặt vấn đề 31 2 Thực nghiệm 34 2.2.1 Hóa chất, thiết bị 34 2.2.2 Tổng hợp vật liệu nano CeO2 34 iii 2.2.3 Tổng hợp vật liệu nano composit CeO2@Ppy 36 2.3 Kết thảo luận 37 2.3.1 Kết tổng hợp vật liệu nano CeO2 37 2.3.2 Kết tổng hợp vật liệu nano composit CeO2@Ppy 47 Kết luận chương 54 Chương 3: PHÁT TRIỂN CẢM BIẾN ADN TRÊN CƠ SỞ CÁC THANH NANO CeO2 56 3.1 Đặt vấn đề 56 Thực nghiệm 59 3.2.1 Hóa chất 59 3.2.2 Cố định ssADN 59 3.2.3 Các phép đo điện hóa 62 3.3 Kết thảo luận 62 3.3.1 Kết cố định chuỗi ssADN 62 3.3.2 Đặc trưng cảm biến ADN 66 3.3.3 Tối ưu hóa điều kiện thực nghiệm 69 3.3.4 Độ lặp lại, độ ổn định, tính chọn lọc khả tái sử dụng 73 Kết luận chương 75 Chương 4: CẢM BIẾN SINH HỌC ADN TRÊN CƠ SỞ VẬT LIỆU NANO COMPOSIT CeO2@Ppy 76 4.1 Đặt vấn đề 76 4.2 Thí nghiệm 78 4.2.1 Hóa chất 78 4.2.2 Cố định chuỗi ssADN dò lên bề mặt điện cực 79 4.2.3 Phân tích điện hóa 79 4.3 Kết thảo luận 79 4.3.1 Kết cố định ssADN 79 4.3.2 Đặc trưng điện hóa cảm biến sinh học ADN 81 4.3.3 Nghiên cứu ảnh hưởng số thơng số đến tín hiệu cảm biến 84 4.3.4 Độ chọn lọc, độ lặp lại độ ổn định cảm biến sinh học ADN 86 iv 4.3.5 Xác định vi khuẩn Salmonella phương pháp PCR so sánh với số liệu phát cảm biến ADN 88 Kết luận chương 89 KẾT LUẬN VÀ KIẾN NGHỊ 90 DANH MỤC CÁC CÔNG TRÌNH ĐÃ CƠNG BỐ CỦA LUẬN ÁN 91 TÀI LIỆU THAM KHẢO 92 v DANH MỤC CÁC KÝ HIỆU VÀ CHỮ VIẾT TẮT TT Ký hiệu, Tên Tiếng Anh chữ viết tắt Tên Tiếng Việt ADN Acid deoxyribonucleic A xít deoxyribonucleic APTES 3-Aminopropyl-triethoxy- 3-Aminopropyl-triethoxy- silane silan ASV Adsortive Stripping Vơn-ampe hịa tan hấp phụ Voltammetry BSA Bovine Serum Albumin Bovine Serum Albumin CE Counter electrode Điện cực đối CPs Conducting Polyme(s) (Các) Polyme dẫn điện CTAB Cetyltrimethyl ammonium cetyltrimetyl amoni bromit bromide CV Cyclic Voltammetry Vơn - ampe vịng DAO Diamin Oxidase Enzym DAO 10 DIG Digoxigenin Digoxigenin 11 DPV Differential Pulse Quét xung vi sai Voltammetry 12 dsADN Double strand ADN Chuỗi ADN kép 13 EDC 1-Ethyl-3-3-Dimethyl- 1-Etyl-3-3-Dimetyl- aminopropyl Carbodiimide aminopropyl Cacbodiimit 14 EDS Energy dispersive spectroscopy 15 EDTA X Ethylene Diamine Tetracetic Etylen Diamin Tetracetic A Acid 16 EIS X-ray Phổ tán sắc lượng tia xít Impedance Phổ tổng trở điện hóa Electrochemical Spectrocopy 17 18 ELISA FESEM Enzym-linked Xét nghiệm miễn dịch liên Immunosorbent assay kết với enzym Field Emission vi Scaning Hiển vi điện tử quét phát xạ trường Electron Microscopy 19 FTIR Fourier Transform Infrared Phổ hồng ngoại biến đổi Spectrocopy 20 hCG Fourier Human Chorionic hooc môn gonadotropin Gonadotropin 21 HRP Horseradish peroxide Horseradish Perơ xít 22 IEP Isoelectric Point Điểm đẳng điện 23 ISFET Ion-Sensitive Field-Effect Trasistor hiệu ứng trường Transistor nhạy ion 24 ITO Indium Tin Oxide Ơ xít thiếc indi 25 LOD Limit of Detection Giới hạn phát 26 LOQ Limit of Quantification Giới hạn định lượng 27 MIA 1-Methylimidazole 1-Metyl imidazol 28 NRs Nanorods Các nano 29 PABA Acid Para-Aminobenzoic A xít 4-Aminobenzoic 30 PANAM Poly Amidoamine Poly amidoamin 31 PANi Polyaniline Polyanilin 32 PBS Phosphat Buffer Saline Đệm phốt phát 33 PCR Polymerase Chain Reaction Phản ứng chuỗi Polyme 34 Ppy Polypyrrole Poly pyrol 35 Py Pyrrole Pyrol 36 RE Reference electrode Điện cực so sánh 37 SCE Saturated Calomel Electrode Điện cực Calomen bão hịa 38 SMOs Solid Metal Oxides xít kim loại bán dẫn 39 ssADN Single strand ADN Chuỗi ADN đơn 40 SWV Square Wave Voltammetry Qt sóng vng 41 TEM Transmission electron Hiển vi điện tử truyền qua microscopy 42 TGA Thermogravimetric Analysis Phân tích nhiệt 43 TMC N-trimetyl chitosan N-trimetyl chitosan vii 44 WE Working electrode Điện cực làm việc 45 XRD X-ray Diffraction Nhiễu xạ tia X viii DANH MỤC CÁC CƠNG TRÌNH ĐÃ CƠNG BỐ CỦA LUẬN ÁN A Tạp chí quốc tế Nguyen Thi Nguyet, Le Thi Hai Yen, Pham Hung Vuong, Phuong Dinh Tam, Tran Trung (2018), “Highly sensitive DNA sensors based on cerium oxide nanorods”, Journal of Physics and Chemistry of Solids, vol 115, pp 18-25 IF: 2.207 Nguyen Thi Nguyet, Vu Y Doan, Nguyen Luong Hoang, Vu Van Thu, Tran Trung, Pham Hung Vuong, Phuong Dinh Tam (2019), “A label-free and highly sensitive DNA biosensor based on the core-shell structured CeO2-NR@ Ppy nanocomposite for Salmonella detection”, Materials Science and Engineering: C, vol 96, pp 790-797 IF: 5.08 Nguyen Thi Nguyet, Vu Van Thu, Hoang Lan, Tran Trung, Anh-Tuan Le, Vuong-Hung Pham, Phuong Dinh Tam (2019), “Simple Label-Free DNA Sensor Based on CeO2 Nanorods Decorated with Ppy Nanoparticles”, Journal of Electronic materials IF: 1.676 B Tạp chí nước Nguyen Thi Nguyet, Tran Trung, Hoang Lan, Vu Van Thu, Pham Hung Vuong, Phuong Dinh Tam (2019), “Direct synthesis of CeO2 nanospindles on gold electrode by electrochemical method”, Vietnam Journal of Chemistry, vol 57, pp 57-63 C Kỷ yếu hội nghị Nguyễn Thị Nguyệt, Lê Thị Hải Yến, Phương Đình Tâm, Trần 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