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Femtosecond Laser-Based Procedures for the Rapid Prototyping Of UNIVERSITÀDEPARTMENT DEGLI OF PHARM SACYTUDI- PH ADIRM BCEUARITIC AAL SCLDOIENC MES ORO DOCTORAL SCHOOL IN PHARMACEUTICAL SCIENCES DIPARTIMENTO INTERATENEO DI FISICA “M. MERLIN" XXXII CYCLE Dottorato di ricerca in FISICA – Ciclo XXXII SSD CHIM/08 Settore Scientifico Disciplinare: FIS/03 Femtosecond laser-based procedures for the rapidDeve loprototypingpment of a fluorescent ofprob epolymeric targeting COX-1 for Lab - fluorescence image-guided surgery (FIGS) in Ovarian Cancer On-a-Chip devices Dottorando: Udith Krishnan Vadakkum Vadukkal PhD Candidate: School Coordinator: Mariaclara Iaselli Prof. Carlo Franchini Supervisori: Supervisor: Dott.Pr ofAntonio. Antonio ScAnconailimati Dott. Ing. Francesco Ferrara Coordinatore: DISSERTATION DEFENSE 2020 Ch.mo Prof. Giuseppe Iaselli ESAME FINALE 2020 2 “To the relentless warriors fighting an incessant battle, To those who dream of ushering in a better world, To my dearest Comrades.” 3 Contents List of abbreviations ............................................................................................. 8 Abstract ................................................................................................................ 10 Introduction …………………………………………………………………….12 Chapter 1 Lab-On-a-Chip …………………...………………………………………14 1.1 Introduction ..................................................................................................... 14 1.2 Lab-on-a-chip for rare cell isolation............................................................. 17 Passive separation and sorting techniques ..................................................... 18 Active separation and sorting techniques ...................................................... 20 1.2.1 LOC techniques for capturing circulating tumour cells (CTC) ................ 21 Microfluidic immunocapture positive enrichment ........................................ 22 1.3 Lab on a chip for in vitro cell cultures ......................................................... 24 Cardiovascular System .................................................................................. 26 Respiratory System ........................................................................................ 27 Digestive and Excretory Systems .................................................................. 28 Nervous system .............................................................................................. 29 1.4 Materials for Lab-On-a-chip ........................................................................ 30 Chapter 2 State of the art of various microfabrication techniques ……………….32 2.1 Introduction ..................................................................................................... 32 2.2 Soft lithography ............................................................................................... 32 2.2.1 Different methods of soft lithography for LOC fabrication ..................... 33 2.2.1.1 Replica mold (REM) technique ......................................................... 33 2.2.1.2 Microcontact print (µCP) technique .................................................. 38 2.2.1.3 Micromolding in capillaries (MIMIC) ............................................... 39 2.2.1.4 Solvent-assisted micromolding (SAMIM) ........................................ 40 2.2.1.5 Micro transfer molding (µTM) .......................................................... 42 2.3 Hot embossing ................................................................................................. 44 2.3.1 Different types of hot embossing ............................................................. 45 2.3.1.1 Plate-to-plate (P2P) hot embossing ................................................... 45 2.3.1.2 Roll-to-plate hot embossing ............................................................... 49 2.3.1.3 Roll-to-roll hot embossing ................................................................. 51 2.3.2 Mold fabrication ....................................................................................... 52 4 2.3.3 Hot embossing for LOC fabrication ......................................................... 53 2.4 Mechanical micro milling ............................................................................... 54 2.4.1 Technical aspects of micro milling .......................................................... 56 2.4.2 Mechanical micro milling for LOC fabrication ....................................... 63 2.5 Femtosecond laser milling .............................................................................. 64 2.5.1 Fs-laser micro-milling: an outstanding ablation procedure ...................... 65 2.5.1.1 Linear absorption ............................................................................... 66 2.5.1.2 Nonlinear absorption ......................................................................... 66 2.5.1.3 Short and ultrashort laser pulses ablation process and timescales ..... 67 2.5.1.4 Ultrashort laser milling ...................................................................... 70 2.5.2 Femtosecond laser ablation for the fabrication of lab-on-a-chips ............ 73 2.6 Bonding of thermoplastic microfluidic devices .............................................. 75 2.6.1 Indirect bonding ....................................................................................... 75 2.6.1.1 Adhesive bonding .............................................................................. 75 2.6.2 Direct bonding .......................................................................................... 77 2.6.2.1 Thermal fusion bonding ..................................................................... 77 2.6.2.2 Solvent bonding ................................................................................. 78 2.6.2.3 Localized welding .............................................................................. 81 2.6.2.4 Wax bonding ...................................................................................... 82 2.6.3 Surface treatments and modifications ...................................................... 83 Chapter 3 Materials and methods …………………………………..…………….…85 3.1 Introduction ..................................................................................................... 85 3.2 Materials ....................................................................................................... 85 3.3 Device fabrication setup ............................................................................... 87 3.3.1 Femtosecond laser micromachining ......................................................... 87 3.3.1.1 Trumpf Laser ..................................................................................... 87 3.3.1.2 Pharos laser ........................................................................................ 89 3.3.2 Mechanical micro-milling ........................................................................ 89 3.3.3 Hot embossing .......................................................................................... 90 3.3.4 Bonding .................................................................................................... 91 3.3.4.1 Isopropanol assisted indirect thermal bonding .................................. 91 3.4 Post-processing analysis equipments .............................................................. 92 3.4.1 Optical microscope ................................................................................... 93 3.4.2 Confocal microscope ................................................................................ 93 5 3.5 Validation setup of the final devices ............................................................... 94 3.5.1 Lab-on-a-chip for circulating tumour cells capturing .............................. 94 3.5.2 Lab-on-a-chip for neuronal cell culturing ................................................ 94 3.5.2.1 Primary hippocampal cell culture ...................................................... 94 3.5.2.2 Immunocytochemistry ....................................................................... 95 Chapter 4 Prediction model of the depth of the femtosecond laser micro-milling of PMMA ………………………………………………………………………………...96 4.1 Introduction ..................................................................................................... 96 4.2 Statistical approach for laser micromachining ................................................ 96 4.3 Experimental procedure .................................................................................. 99 4.4 Results and discussions ................................................................................. 102 4.4.1 Full Factorial plan .................................................................................. 102 4.4.2 Validation of prediction model ............................................................... 108 Chapter 5 Smart procedure for the femtosecond laser-based fabrication of polymeric lab-on-a-chip for tumor cells capturing ……………………………….110 5.1 Introduction ................................................................................................... 110 5.2 Fabrication of LOC device ............................................................................ 111 5.3 Bonding and functionalization of the device ................................................ 113 5.4 CTC capture experiments .............................................................................. 114 5.5 Discussion ....................................................................................................
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