Biomedical Engineering Technologies : Volume 2

Dedication Preface Biosensing Modalities References Contents Contributors Part I: Molecular and Cellular Analysis and Manipulation Chapter 1: Development of a Multi-target Protein Biomarker Assay for Circulating Tumor Cells 1 Introduction 2 Materials 2.1 Device Fabrication 2.2 Working Solutions 2.3 Tumor Cell Culture 2.4 Equipment 2.5 Reference 600 Potentiostat Gamry Instrument 3 Methods 3.1 Bottom Gold Electrode Fabrication 3.2 Top Electrode Fabrication 3.3 Bottom Gold Electrode Functionalization 3.4 CNT Functionalization 3.5 Fabrication of Membrane 3.6 Package of Filtration Device 3.7 Tumor Cells Collection 3.8 Tumor Cells Spiked in Human Blood Sample Lysate 3.9 Shearing Platform 3.10 Shear Optimization 3.11 Detection of Tumor Cells Spiked in Blood Samples 4 Notes References Chapter 2: Method to Isolate Dormant Cancer Cells from Heterogeneous Populations 1 Introduction 2 Materials 2.1 Protocol 1: Silica-Polyethylene Glycol (SPEG) Gel Immobilization 2.2 Protocol 2: Agarose Pre-coating Followed by Silica Gel (SPEG10K) Immobilization 3 Methods 3.1 Protocol 1: Silica-Polyethylene Glycol (SPEG) Gel Immobilization 3.1.1 Prepare Cell Solution 3.1.2 Prepare Gelling Solution and THEOS 3.1.3 Encapsulate Cells in SPEG Gel 3.2 Protocol 2: Agarose Pre-Coating Followed by Silica Gel (SPEG10K) Immobilization 3.2.1 Microfluidic Flow Focusing Device 3.2.2 Emulsion Oil 3.2.3 Gelling Solution 3.2.4 Prepare THEOS 3.2.5 Cell Solution 3.2.6 Microfluidic Setup 3.2.7 Agarose Coating of Cells 3.2.8 Cell Encapsulation in SPEG10K 3.2.9 Cell Collection and Isolation from SPEG10K 4 Notes References Chapter 3: Label-Free Morphological Phenotyping of In Vitro 3D Microtumors 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Cantilevers 2.3 Image Acquisition and Analysis 3 Methods 3.1 Preparation of Multicellular Spheroids 3.1.1 Preparation of Tumor Spheroids 3.1.2 Preparation of Tumor-Fibroblast Co-culture Spheroids 3.2 Fabrication of Cantilevers 3.3 Microindentation and 4D Image Acquisition 3.4 Deformation Analysis 4 Notes References Chapter 4: High-Throughput Microenvironment Microarray (MEMA) High-Resolution Imaging 1 Introduction 2 Materials 3 Methods 3.1 MEMA 3.2 Imaging (see Note 2 and Note 3) 3.3 Navigating 4 Notes References Chapter 5: Real-Time Analysis of AKT Signaling Activities at Single-Cell Resolution Using Cyclic Peptide-Based Probes 1 Introduction 2 Materials 2.1 Liposomes 2.2 Dye-Conjugated Polystyrene Beads 2.3 Microwell Chip 2.4 Cell Lines 2.5 Immunofluorescence 3 Methods 3.1 Microwell Single-Cell Chip Fabrication 3.2 Preparation of Liposomes 3.3 Synthesis of Dye-Conjugated Polystyrene Beads 3.4 Cell Loading 3.5 Confocal Imaging of Living Cells 3.6 Immunofluorescence 3.7 Data Extraction and Correction 3.8 Dynamic Time Warping and Clustering Analysis of the Single-Cell Data 4 Notes References Chapter 6: Microfluidic Device Technologies for Digestion, Disaggregation, and Filtration of Tissue Samples for Single Cell Ap... 1 Introduction 2 Materials 3 Methods 3.1 Digestion Device Fabrication 3.2 Dissociation Device Fabrication 3.3 Filter Device Fabrication 3.4 Tissue Preparation 3.5 Digestion Device Setup and Operation 3.6 Dissociation Device Setup and Operation 3.7 Filter Device Setup and Operation for Direct Flow Mode 3.8 Filter Device Setup and Operation for Tangential Flow Mode 4 Notes References Chapter 7: Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine 1 Introduction 2 Materials 2.1 Immunohistochemistry (IHC) Materials 2.2 Laser Capture Microdissection (LCM) Materials 2.3 ddPCR Materials 3 Methods 3.1 Pathology Review 3.2 Immunohistochemistry (IHC) General Procedure 3.3 Immuno-LCM 3.4 DNA Extraction, Quality Assurance, and Preparation for ddPCR 3.5 ddPCR Assay Setup 3.6 ddPCR Analysis and Reporting 3.7 Conclusions 4 Notes References Chapter 8: Functionalized Lineage Tracing for the Study and Manipulation of Heterogeneous Cell Populations 1 Introduction 2 Materials 2.1 Equipment 2.2 Disposables 2.3 Biologics 2.4 Plasmids 2.5 Primers 2.6 Buffers 2.7 Enzymes 2.8 Other Reagents 2.9 Computational 3 Methods 3.1 sgRNA Barcode Library Plasmid Pool Assembly 3.2 sgRNA Barcode Sampling 3.3 sgRNA Barcoding Lentivirus Production 3.4 Determine sgRNA Viral Titer 3.4.1 Titering on Adherent Cells (Forward Procedure) 3.4.2 Titering on Suspension Cells (Reverse Procedure) 3.4.3 Flow Cytometry to Determine Viral Titer 3.5 sgRNA Barcode Transduction 3.6 Targeted sgRNA Barcode Sampling of Cells 3.6.1 Preparing Samples for Sequencing 3.6.2 Processing Barcode Sequencing Data 3.6.3 Processing CROP-Seq Barcodes from 10x Cell Ranger Output 3.7 Recall Plasmid Assembly 3.8 Recall and Isolation of Barcoded Lineages 4 Notes References Chapter 9: Fluorescence Lifetime Imaging Probes for Cell-Based Measurements of Enzyme Activity 1 Introduction 1.1 Biosensors for Kinases and Cell-Based Activity Analyses 1.1.1 Kinases in Cancer 1.1.2 Methods for Kinase Activity Detection 1.1.3 Fluorescence Lifetime Imaging Probe Technology 1.1.4 Probe Design Consideration Peptide Substrate Selection Fluorophore Selection and Labeling 1.2 Fluorescence Lifetime Analyses 1.2.1 Fluorescence Lifetime and Decay 1.2.2 Advantages of Lifetime Imaging over Steady State Measurements 1.2.3 Time Domain Acquisition 1.2.4 Frequency Domain Acquisition 1.3 Imaging Hardware Requirements 1.3.1 Pulsed Light Sources, for Example 1.3.2 Appropriate Microscopic Optics 1.3.3 Single Photon Detection Modules with Appropriate Sensitivity and Time Resolution 1.4 Experimental Design for Fluorescence Lifetime-Based Kinase Activity Measurements 1.4.1 Peptide Sequences 1.4.2 Fluorophore Labels. 1.4.3 Cell Culture Consumables for Cellular Imaging 1.4.4 Positive and Negative Controls Synthetic Peptide Controls for Fluorescence Lifetime Signal Validation Biological Controls 1.4.5 Biological Conditions Cell Culture Conditions Cell Preparation and Biosensor Incubation Kinase Activation 1.5 Lifetime Image Acquisition Parameters 1.5.1 Detection Rate 1.5.2 Pulsing Frequency 1.5.3 IRF Response 1.5.4 Photon Economy 1.5.5 Background Signal Considerations 1.5.6 Photobleaching 1.5.7 Multiplexing Multiplexing with Time Course Experiments 1.6 Data Analysis 1.6.1 Lifetime Fitting Dissecting Different Signal Components and Background Relative Quantification of Phosphorylated Probe 1.6.2 Image Reconstruction for Data Visualization 2 Materials 2.1 Imaging Hardware/Software 2.2 Chemicals and Supplies 3 Methods (See Note 1) (Fig. 7) 4 Notes References Chapter 10: Assessment of Intracellular GTP Levels Using Genetically Encoded Fluorescent Sensors 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Transfection Reagents 2.3 Microscopy 2.4 Software for Analysis 3 Methods 3.1 Lentiviral Supernatant Preparation and Delivery to Target Cells 3.2 Preparation of Cells for Imaging 3.3 Cell Imaging 3.4 Images Analysis 4 Notes References Chapter 11: Node-Pore Sensing for Characterizing Cells and Extracellular Vesicles 1 Introduction 2 Materials 2.1 NPS Measurement Platform 2.1.1 Custom Printed Circuit Board (PCB) 2.1.2 Shielded Box and Accessories 2.1.3 Equipment and Software 2.2 NPS Devices 2.3 Consumables 2.4 Sample and Sample Preparation 3 Methods 3.1 NPS Device Fabrication 3.1.1 Electrode Fabrication 3.1.2 SU-8 Master Fabrication (Exo-NPS) 3.1.3 SU-8 Master Fabrication (mNPS) 3.1.4 Soft Lithography and Device Assembly 3.2 Assembly and Setup of NPS Measurement Platform (Fig. 6) 3.3 Node-Pore Sensing for Detection of EVs Displaying Specific Surface Markers (Fig. 4) 3.4 Node-Pore Sensing for Mechanical Phenotyping of Cells (Fig. 5) 4 Notes References Chapter 12: Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes 1 Introduction 2 Materials 2.1 Preparation of Nanostructured Substrates 2.2 LNP Surface Immobilization 2.3 Micromixer Fabrication and Device Assembly 2.4 EV Isolation 2.5 Detection of EV Protein Marker and Mutations in EV DNA 3 Methods 3.1 Fabrication of Nanostructured Substrates and LNP Immobilization 3.2 Micromixer Device Assembly 3.3 EV Isolation 3.4 Detection of EV Protein Markers and Mutation in EV DNA with ddPCR 4 Notes (Denoted as Subscript) References Chapter 13: Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR 1 Introduction 2 Materials 2.1 MSP Assay Chip 2.2 Heater, Magnetic Support, and Fluorescent Detection 2.3 DNA Isolation, Bisulfite Conversion, and PCR Reagents 2.4 Fluorescence Detection 3 Methods 3.1 Chip Fabrication 3.2 Reagent Loading 3.3 Assay Procedure 3.4 qPCR Detection 4 Notes References Chapter 14: Droplette: A Platform Technology to Directly Deliver Nucleic Acid Therapeutics and Other Molecules into Cells and ... 1 Introduction 1.1 Characteristics of Droplette Output 1.2 Summary of Studies Done Using Droplette 1.3 Droplette Delivery Deep into Cells and Tissues 1.3.1 Example Application 1: Transfecting Adherent Cell Cultures Using Droplette 1.3.2 Example Application 2: In Vivo Droplette Delivery of DNA in Mice 2 Materials 2.1 Droplette Device Fixture (with Components Necessary to Build Out in Different Forms) 2.2 Reagents for Transfection in Cells and Animals 3 Methods 3.1 Droplette Device Overview 3.2 Droplette Device Assembly 3.3 Sample Preparation for Use with Droplette 3.4 Quantifying Device Performance 3.5 Verifying Functionality of Biomolecules That Pass Through Droplette System 3.6 Example Use Case 1: Transfecting Adherent Cell Cultures Using Droplette 3.6.1 Seed Adherent Cells in Petri Dishes 3.6.2 Prepare DNA Master Mix for DNA-Receiving Samples 3.6.3 Negative Control 3.6.4 Droplette Delivery 3.6.5 Incubation and Analysis 3.7 Example Use Case 2: In Vivo Droplette Delivery of Plasmid DNA into Mice 3.7.1 Shave and Clean Delivery Site as Needed 3.7.2 Stratum Corneum (SC) Removal as Needed 3.7.3 Droplets Delivery 3.7.4 Mouse Recovery 3.7.5 Incubation and Analysis 4 Notes References Chapter 15: Molecular Imaging of HER2 in Patient Tissues with Touch Prep-Quantitative Single Molecule Localization Microscopy 1 Introduction 2 Materials 2.1 Coverslip Preparation 2.2 Touch Prep 2.3 Sample Preparation 2.3.1 Antibody and Fluorescent Dye Conjugation 2.3.2 Immunohistochemistry 2.4 dSTORM Imaging 2.5 Data Analysis 3 Methods 3.1 Coverslip Preparation 3.2 Touch Prep 3.3 Sample Preparation 3.3.1 Antibody and NHS Fluorescent Dye Conjugation 3.3.2 Immunohistochemistry 3.4 dSTORM Sample Imaging 3.5 Data Processing 3.6 Data Analysis 3.6.1 Localization Density Filter 3.6.2 Average Number of Localizations for Trastuzumab-AF647 3.6.3 Protein Auto-Correlation Analysis 3.6.4 Cluster Occupancy Analysis 3.6.5 Correlation with Clinical Data 4 Notes References Chapter 16: Microchip Free-Flow Electrophoresis for Bioanalysis, Sensing, and Purification 1 Introduction 1.1 Free-Flow Electrophoresis 1.2 Principles of Microfluidics and Microfluidic Device Design 2 Materials 2.1 Photolithography 2.2 PDMS Device Fabrication 2.3 Device Operation and Imaging 2.4 Solutions and Reagents 3 Methods 3.1 Design of Microfluidic Devices 3.2 Photolithographic Fabrication of Device Master 3.3 Fabrication of PDMS Devices 3.4 Device Priming and Preparation 3.5 On-Chip Fractionation of Biological Mixtures 4 Notes References Chapter 17: Green Chemistry Preservation and Extraction of Biospecimens for Multi-omic Analyses 1 Introduction 2 Materials 2.1 Stock Liver Tissues 2.2 Lipidomics 2.3 Transcriptomics and Metabolomics 2.4 Genomics 2.5 Proteomics 3 Methods 3.1 Collection and Processing of Stock Mouse Liver 3.2 Collection and Processing of Stock Human Liver 3.3 Lipidomics: Step 1 in Fig. 3 3.3.1 CIPS: Steps 1 and 2 in Fig. 3 3.3.2 Folch 3.3.3 NMR Sample Preparation and Analysis 3.3.4 LC-MS Sample Preparation and Analysis 3.4 Transcriptomics: Step 2 in Fig. 3 3.5 Metabolomics: Step 3 in Fig. 3 3.5.1 Acetonitrile Extraction 3.5.2 Perchloric Acid Extraction 3.5.3 Methanol Extraction 3.5.4 NMR Spectroscopy Determination of Acetonitrile, Methanol, Perchloric Acid Extraction Efficiencies 3.6 Proteomic: Step 4 in Fig. 3 3.6.1 Protein Extraction 3.6.2 Standard Digestion of Protein Sample 3.6.3 Solid-Phase Extraction Desalting 3.6.4 nanoLC-MS Proteomic Analysis 3.7 Genomics: Step 5 in Fig. 3 3.7.1 Use Entire Pellet from the TRIzolTM Extraction Step Above (Subheading 3.6.1, step 9) 4 Notes References Chapter 18: TdT-dUTP DSB End Labeling (TUDEL), for Specific, Direct In Situ Labeling of DNA Double Strand Breaks 1 Introduction 2 Materials 2.1 Sample and Slide Preparation 2.2 TUDEL Staining and Immunofluorescence 2.3 Imaging and Data Analysis 3 Methods 3.1 Cell Culture and Sample Preparation 3.2 DNA Damage Induction 3.3 Fixation and Slide Preparation 3.4 TUDEL 3.5 Immunostaining 3.6 Imaging and Quantification of DSBs 3.7 Superresolution Imaging of DSBs (Optional) 4 Notes References Chapter 19: Ligand-Directed GPCR Antibody Discovery 1 Introduction 1.1 Library Construction, Screening, and Validation: Macrocycle 1 1.2 Library Construction, Screening, and Validation: Macrocycle 2 2 Material 3 Methods 3.1 First-Generation Library Design and Construction. Oligo Design 3.1.1 Annealing 3.1.2 Kunkel Heteroduplex Formation 3.2 Bulk Panning 3.3 Negative Panning (Round 1) 3.4 Sorting/Positive Selection 3.5 Preliminary Affinity Analysis Using Flow Cytometry 3.6 GPCR Functional Assays 3.7 Affinity Maturation Library Construction, Panning, and Validation 3.8 Bio-panning and Validation 4 Notes References Chapter 20: Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immuno... 1 Introduction 2 Materials 2.1 Nanosensor Fabrication 2.2 Flow Cell Fabrication 2.3 Optical System 2.4 Electrical System 2.5 Protein Solutions 3 Methods 3.1 Nanosensor Fabrication 3.1.1 SANE Sensor Chip Design 3.1.2 Fabrication of a Multi-layer Free-Standing Metal-Dielectric Membrane 3.1.3 Milling of Double Nanohole-Nanopore Structures 3.2 Flow Cell Fabrication 3.3 Experimental Setup 3.3.1 Optical Sensing System 3.3.2 Sample Loading 3.3.3 Placement of Flow Cell 3.3.4 Electrical Sensing System 3.3.5 Finding the Double Nanohole (DNH) 3.4 Data Acquisition: The Bimodal Optical and Electrical Data Types 3.5 Detection of Biomolecular Interactions 3.5.1 Generation of Antigens and Antibodies 3.5.2 Sample Preparation 3.5.3 Using Bimodal Data Types to Differentiate Between Antibody and Ligand 3.5.4 Determining Threshold Values to Distinguish Bound Complexes from Unbound Molecules 3.5.5 Distinguishing Between Specific and Non-specific Ligand-Antibody Interactions 4 Notes References Chapter 21: Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast 1 Introduction 2 Materials 2.1 Site-Specific Incorporation of ncAAs in Proteins in Yeast 2.2 Flow Cytometry- and Microplate Reader-Based Evaluation of ncAA Incorporation Events in Yeast 2.3 Bioorthogonal Reactions with ncAAs on the Yeast Surface 2.4 Click Chemistry Analysis: Flow Cytometry and Extent of Reaction Calculations 2.5 Preparation of Libraries Involving the Use of Orthogonal Translation Systems 3 Methods 3.1 Site-Specific Incorporation of ncAAs in Proteins in Yeast 3.1.1 Preparation of RJY100 Cells Prior to Preparing Chemically Competent Yeast (Fig. 2a) 3.1.2 Preparation of Chemically Competent Yeast (See Note 39) 3.1.3 Transformations of pPOIVector and pOTSVector in Chemically Competent Yeast (Fig. 2b) 3.1.4 Preparation of RJY100 Control Samples (See Note 44) 3.1.5 Yeast Propagation in Liquid Media (Fig. 2c) 3.1.6 Dilution of Yeast Cultures to OD600 of 1 (Fig. 2c) 3.1.7 Yeast Inductions (Fig. 2c) 3.2 Flow Cytometry- and Microplate Reader-Based Evaluation of ncAA Incorporation Events in Yeast 3.2.1 Yeast Preparation for Flow Cytometry: Primary Labeling of Displayed Proteins (Fig. 4a) 3.2.2 Yeast Preparation for Flow Cytometry: Secondary Labeling of Displayed Proteins (Fig. 4a) 3.2.3 Yeast Preparation for Flow Cytometry: No Labeling Required (For Intracellular Fluorescent Protein Detection, Fig. 4b) 3.2.4 Flow Cytometry with Displayed or Intracellular ncAA-Containing Reporters: Collection Settings, Controls, and Data Collec... 3.2.5 Yeast Preparation for Microplate Reader Assays (Fig. 4c) 3.2.6 Microplate Reader Protocols for Intracellular Reporters 3.2.7 Flow Cytometry Data Analysis for RRE and MMF (Fig. 5) 3.2.8 RRE and MMF Metrics from Flow Cytometry (Worksheet, Sheet 1: Flow RRE MMF; Fig. 3) 3.2.9 Single-Fluorescent Protein ncAA Incorporation Efficiency (Fraction WT) and Misincorporation (Misincorporation) Metrics f... 3.2.10 Dual-Fluorescent Protein ncAA Incorporation RRE and MMF Metrics from a Microplate Spectrophotometer (Worksheet, Sheet 3... 3.2.11 Single-Fluorescent Protein ncAA Incorporation Efficiency (Fraction WT) and Misincorporation (Misincorporation) Metrics ... 3.3 Bioorthogonal Reactions with ncAAs on the Yeast Surface 3.3.1 Preparation of Induced Cells for Click Chemistry Reactions 3.3.2 One-Step Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Click Chemistry on the Yeast Surface (Fig. 6a) 3.3.3 Two-Step CuAAC Click Chemistry on the Yeast Surface: Reaction with Desired Cargo (Step 1) (Fig. 6b) 3.3.4 Two-Step CuAAC Click Chemistry on the Yeast Surface: Reaction with Biotin Probe (Step 2) (Fig. 6b) 3.3.5 One-Step Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) Click Chemistry on the Yeast Surface (Fig. 6a) 3.3.6 Two-Step Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) Click Chemistry on the Yeast Surface: Reaction with Desired ... 3.3.7 Two-Step Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) Click Chemistry on the Yeast Surface: Reaction with Biotin P... 3.4 Click Chemistry Analysis: Flow Cytometry and Extent of Reaction Calculations 3.4.1 Yeast Preparation for Flow Cytometry (Labeling) 3.4.2 Flow Cytometry: Collection Settings, Controls, and Data Collection 3.4.3 Flow Cytometry Data Analysis for Click Chemistry Extent of Reaction 3.4.4 Click Chemistry Extent of Reaction Analysis (Worksheet, Sheet 5: Extent of Reaction; Fig. 6c) 3.5 Preparation of Libraries Involving the Use of Orthogonal Translation Systems 3.5.1 DNA Preparation of an Orthogonal Translation System Library with a ncAA Incorporation Protein Reporter: Vector Digest (F... 3.5.2 DNA Preparation of a Yeast-Displayed Protein Library with an Orthogonal Translation System: Vector Digest (Fig. 7b) 3.5.3 DNA Preparation for Electroporation: DNA Amplification via PCR (Fig. 7a, b) 3.5.4 DNA Preparation for Electroporation: Gel Extractions and DNA Purification (Fig. 7a, b) 3.5.5 DNA Preparation for Electroporation: Pellet Paint Procedure for Libraries of OTSs (Fig. 7a) 3.5.6 DNA Preparation for Electroporation: Pellet Paint Procedure for Libraries of POIs (Fig. 7b) 3.5.7 Preparation of RJY100 Cells Containing pPOIVector-BXG Prior to Electroporation for Use with Libraries of OTSs (Fig. 7a, ... 3.5.8 Preparation of RJY100 Cells Containing pRS315-LeuOmeRS Prior to Electroporation for Use with Libraries of Yeast-Displaye... 3.5.9 Preparation of Electrocompetent RJY100 (Fig. 7c) 3.5.10 Electroporation of DNA in Electrocompetent RJY100 (Fig. 7c) 3.5.11 Library Characterization: Transformation Efficiency (Fig. 7c) 3.5.12 Library Propagation and Storage (Fig. 7c) 3.5.13 Library Characterization: Flow Cytometry (Fig. 7c) 3.5.14 Library Characterization: Yeast Minipreps for DNA Sequencing (Fig. 7c, See Note 77) 3.5.15 Library Characterization: E. coli Transformations for DNA Sequencing (Fig. 7c) 4 Notes References Chapter 22: Nuclease-Assisted, Multiplexed Minor-Allele Enrichment: Application in Liquid Biopsy of Cancer 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 23: Implementation of Ion Mobility Spectrometry-Based Separations in Structures for Lossless Ion Manipulations (SLIM) 1 Introduction 2 Materials 2.1 Electrospray Ionization Source 2.2 High-Pressure Ion Optical System Before SLIM 2.3 TW-SLIM Modules 2.4 Low Pressure Ion Optical System After SLIM 2.5 Data Acquisition 3 Methods 3.1 Ion Formation 3.2 Connecting Voltages to SLIM 3.3 Transmission Mode: Tuning for Signal Intensities of Analyte(s) of Interest 3.4 Ion Accumulation 3.5 Parameter Optimization for Resolution and Sensitivity in Separation (IMS) Mode 3.6 Ion Switch for Multipass Separations 3.7 Data Processing 4 Notes References Chapter 24: Pleural Effusion Aspirate for Use in 3D Lung Cancer Modeling and Chemotherapy Screening 1 Introduction 2 Materials 2.1 Cell Culture of Effusion Aspirate 2.2 Cell Culture in 3D 2.3 Drug Screening 2.4 Phenotypic Observation and Quantification 3 Methods 3.1 Cell Isolation from Pleural Effusion Aspirate 3.2 Preparation for Cell Culture in 2D and 3D 3.3 Cell Culture in 2D Dishes 3.4 3D Organoid Culture 3.5 Phenotypic Observation and Quantification 3.6 Drug Screening and Quantification 3.6.1 2D Cell Culture 3.6.2 3D Organoid Culture 4 Notes References Chapter 25: Using Optical Tweezers to Dissect Allosteric Communication Networks in Protein Kinases 1 Introduction 2 Materials 2.1 Optical Tweezer Apparatus 2.2 Microfluidic Chamber 2.3 Beads Preparation 2.4 Sample Preparation for Optical Tweezers Samples 2.5 Oligo Sequence for Double-Stranded DNA Handles 3 Methods 3.1 Microfluidic Chambers 3.2 Bead Preparation 3.3 Sample Preparation for Optical Tweezers Measurement 3.3.1 Protein Construct Design 3.3.2 Integrated Method to Attach dsDNA Handles and Select Functional Proteins 3.4 Optical Tweezers Measurement 3.5 Example of Results 4 Notes References Part II: Therapeutics Technologies Chapter 26: Focused Ultrasound-Mediated Intranasal Brain Drug Delivery Technique (FUSIN) 1 Introduction 2 Materials 2.1 Intranasal Delivery 2.2 FUS Treatment 3 Methods 3.1 . Intranasal Administration 3.2 Focused Ultrasound Treatment 4 Notes References Chapter 27: Extracellular pH Mapping as Therapeutic Readout of Drug Delivery in Glioblastoma 1 Introduction 2 Materials 2.1 In Vitro Calibration of TmDOTP5- pH Sensitivity 2.2 Animal Preparation for In Vivo Tumor Studies 2.3 Animal Setup for Magnetic Resonance Imaging/Spectroscopy Experiments 2.4 Contrast Agent Preparation 2.5 MRI and BIRDS Acquisition 2.6 MRI and BIRDS Analysis 2.7 Treatment Studies 3 Methods 3.1 In Vitro Calibration of TmDOTP5- pH Sensitivity 3.2 Animal Preparation for In Vivo Tumor Studies 3.3 Animal Setup for Magnetic Resonance Imaging/Spectroscopy Experiments 3.4 Contrast Agent Preparation 3.5 MRI and BIRDS Acquisition 3.6 MRI and BIRDS Analysis 3.7 Treatment Studies 4 Notes References Chapter 28: Charge-Based Multiarm Avidin Nanoconstruct as a Platform Technology for Applications in Drug Delivery 1 Introduction 2 Materials 2.1 Biotinylation of Eight-Arm PEGs 2.2 Synthesis of Ester Linkers to Conjugate Dexamethasone (Dex) with Biotinylated PEG 2.3 Synthesis of Multiarm Avidin (mAv) Nanoconstruct 2.4 Bioefficacy of mAv-Dex Compared with Dex Using In Vitro Cytokine Challenged Cartilage Explant Culture Model of Osteoarthri... 3 Methods 3.1 Biotinylation of Eight-Arm PEGs (PEG-Biotin) 3.2 Synthesis of Ester Linkers to Conjugate Dex with PEG-Biotin 3.2.1 Synthesis of Dexamethasone Hemisuccinate (Dex-SA), Glutarate (Dex-GA), and Phthalate (Dex-PA) 3.2.2 Conjugation of Dex-SA, Dex-GA, and Dex-PA (Compounds 2, 3, 4) to PEG-Biotin 3.3 Synthesis of (Multiarm Avidin) mAv Nanoconstruct 3.4 Bioefficacy of mAv-Dex Compared with Dex Using In Vitro Cytokine Challenged Cartilage Explant Culture Model of Osteoarthri... 4 Notes References Chapter 29: Chemical Modification of Proteins and Their Intracellular Delivery Using Lipidoid Nanoparticles 1 Introduction 1.1 Drugging the Undruggable 1.2 Challenges with Intracellular Delivery of Protein Therapeutics 1.3 Approaches for Intracellular Delivery of Proteins 1.4 Protein Modification and Intracellular Delivery 2 Materials 2.1 NBC Modification of Protein and Its Intracellular Delivery 2.2 Aco Modification of Protein and Its Intracellular Delivery 2.3 HA Modification of Protein and Its Intracellular Delivery 2.4 Instruments and Software 3 Methods 3.1 NBC Modification of Protein and Its Intracellular Delivery 3.1.1 Conjugation and Validation of RNase A-NBC 3.1.2 Intracellular Delivery of RNase A-NBC Enabled by Cationic Lipidoid Nanoparticles 3.2 Aco Modification of Protein and Its Intracellular Delivery 3.2.1 Conjugation and Validation of RNase A-Aco 3.2.2 Intracellular Delivery of Aco-Conjugated Proteins Enabled by Lipidoid Nanoparticles 3.3 HA Modification of Protein and Its Intracellular Delivery 3.3.1 Conjugation and Validation of RNase A-HA 3.3.2 Intracellular Delivery of RNase A-HA Enabled by EC16-80 Lipidoid Nanoparticles 4 Notes References Chapter 30: Generation of Membrane-Derived Nanovesicles by Nitrogen Cavitation for Drug Targeting Delivery and Immunization 1 Introduction 2 Materials 2.1 Strains 2.2 Reagents and Kits 2.3 Working Solutions 2.4 Instruments 3 Methods 3.1 Generation of Cell Membrane-Derived Nanovesicles for Endothelium Targeting 3.1.1 Preparation of Cells 3.1.2 Preparation of EVs 3.1.3 Hydrophobic Drug Loading to Nanovesicles 3.1.4 Weak Acid Drug Remote Loading Inside Nanovesicles (HL 60 Cell-derived EVs) 3.2 Application of EVs in the Therapy for Inflammation 3.2.1 Enhanced Therapy of EVs for Acute Lung Inflammation (ALI) 3.2.2 Enhanced Therapy of EVs for Peritonitis 3.3 Generation of Bacterium Membrane-Derived Nanovesicles 3.3.1 Preparation of Bacteria 3.3.2 DMV Preparation by Nitrogen Cavitation 3.4 Application of DMVs as a Vaccine 4 Notes References Chapter 31: Laboratory-Scale Production of Sterile Targeted Microbubbles 1 Introduction 2 Materials 3 Methods 3.1 Material Sterilization 3.2 Preparation of Sterilized Microbubbles (MBs) 3.3 MB-Antibody Conjugation 3.4 MB Characterization 4 Notes References Chapter 32: Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces 1 Introduction 2 Materials 2.1 Immobilization of AAV2 Vectors on a Stainless-Steel Substrate 2.2 Fluorescent Labeling of AAV2 and Substrate Immobilization of Fluorescent AAV2 Particles 2.3 Immunofluorescence Staining of AAV2 Immobilized on the Surface of Metal Substrate 2.4 Scanning Electron Microscopy of AAV2-Laden Stainless-Steel Surfaces 2.5 Quantification of Vector Load with RT-PCR 2.6 Cell Transduction with Mesh Disk-Immobilized AAV2-eGFP Particles 2.7 Preparation and Deployment of AAV2-Eluting Stents in the Rat Carotid Model 2.8 Bioluminescence Imaging of Arterial Gene Expression 3 Methods 3.1 Immobilization of AAV2 Vectors on a Stainless-Steel Substrate (Fig. 1) 3.2 Fluorescent Labeling of AAV2 and Substrate Immobilization of Fluorescent AAV2 Particles (Fig. 2) 3.3 Immunofluorescence Staining of AAV2 Immobilized on the Surface of Metal Substrate (Fig. 3) 3.4 Scanning Electron Microscopy of AAV2-Laden Stainless-Steel Surfaces (Fig. 4) 3.5 Quantification of Vector Load with RT-PCR 3.6 Cell Transduction with Mesh Disk-Immobilized AAV2-eGFP Particles (Fig. 5) 3.7 Preparation and Deployment of AAV2-Eluting Stents in the Rat Carotid Model 3.8 Bioluminescence Imaging of Arterial Gene Expression (Fig. 6) 4 Notes References Chapter 33: Decellularization and Recellularization Methods for Avian Lungs: An Alternative Approach for Use in Pulmonary Ther... 1 Introduction 2 Materials 2.1 Avian Lung Harvesting 2.2 Decellularization Process 2.3 Histological Assessment 2.4 Residual DNA and Detergent Assessment 2.5 Electron Microscopy 2.6 Immunohistochemical Staining 2.7 Mass Spectrometry 2.8 Artificial Pleura for Avian Lungs 2.9 Recellularization with Human Lung Cells 3 Methods 3.1 Working Area Setup 3.2 Chicken Lung Harvesting 3.3 Emu Lung Harvesting 3.4 Decellularization Process 3.5 Assessing Structure and Histology 3.6 Residual DNA and Detergent Assessment 3.7 Immunohistochemical Staining 3.8 Mass Spectrometry 3.9 Artificial Pleura for Avian Lungs 3.10 Recellularization Techniques with Human Lung Cell Types 4 Notes References Chapter 34: Methods for Forming Human Lymphatic Microvessels In Vitro and Assessing their Drainage Function 1 Introduction 2 Materials 2.1 Fabrication of Microfluidic Chambers 2.2 Endothelial Cell Culture 2.3 Formation of Lymphatic Microvessels 2.4 Measurement of Drainage Rates 3 Methods 3.1 Fabrication of Microfluidic Chambers 3.2 Endothelial Cell Culture 3.3 Formation of Lymphatic Microvessels 3.3.1 Formation of Collagen Gels that Contain a Blind-Ended Channel 3.3.2 Formation of Blind-Ended Lymphatics in Patterned Gels 3.4 Measurement of Drainage Rates 3.4.1 Measurement of Fluid Drainage Rates 3.4.2 Measurement of Solute Drainage Rates 4 Notes References Chapter 35: Natural Polymer-Based Micronanostructured Scaffolds for Bone Tissue Engineering 1 Introduction 2 Materials 2.1 Preparation of Microspheres 2.2 Preparation of Microporous Sintered Microsphere Scaffolds 2.3 Preparation of Micronanostructured Scaffolds 2.4 Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) 2.5 Scanning Electron Microscopy 2.6 Collagen Content Quantification 2.7 Confocal Microscopy of Live and Dead Cells/Cell Proliferation/Calcium Deposit Quantification/Immunohistochemistry 3 Methods 3.1 Fabrication of Microspheres 3.1.1 Background Reading 3.1.2 Procedure 3.2 Fabrication of 3D Microporous Sintered Matrices 3.2.1 Background Reading 3.2.2 Procedure 3.3 Fabrication of Collagen Nanofibers 3.3.1 Background Reading 3.3.2 Procedure 3.4 Microsphere Surface Chemistry Using ATR-FTIR Analysis 3.4.1 Background Reading 3.4.2 Procedure 3.5 Collagen Content Quantification 3.5.1 Procedure 3.6 Scanning Electron Microscopy 3.6.1 Background 3.6.2 Procedure 3.7 Cell Culture Studies 3.7.1 Background Reading 3.7.2 Procedure 3.8 Live/Dead Assay-Cell Viability 3.8.1 Background Reading 3.8.2 Procedure 3.9 Cell Proliferation 3.9.1 Background Reading 3.9.2 Procedure 3.10 Alkaline Phosphatase Activity 3.10.1 Background Reading 3.10.2 Procedure 3.11 Alizarin Red Assay 3.11.1 Background 3.12 Procedure 3.13 Immunohistochemistry 3.13.1 Background 3.13.2 Procedure 3.14 Statistical Analysis 4 Notes References Chapter 36: Biodegradable Electrospun Nanofibrous Scaffolds for Bone Tissue Engineering 1 Introduction 1.1 Proc