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 Process Mechanism
2 Materials
2.1 Basic Setup Apparatus
2.2 Chemicals
3 Methods
3.1 Apparatus Construction
3.2 Basic Strategies of Electrospinning
3.2.1 Solution Preparation.
Synthesis of Hydroxyapatite (HA) Nanoparticles.
Preparation of Composite Solution
3.2.2 Electrospinning Process for the Fabrication of Electrospun Nanofibrous Scaffold
Fluidics
Electrospun Settings
3.3 Electrospinning Parameters
3.4 Solution Parameters
3.5 Processing Parameters
3.6 Fabrication Techniques
4 Notes
References
Chapter 37: Biotribometer for Assessment of Cell and Tissue Toxicity of Orthopedic Metal Implant Debris
1 Introduction
2 Materials
2.1 Biotribometer
2.2 Test Materials
2.3 Laboratory Equipment
3 Methods
3.1 Test Preparation and Start
3.2 Test End
3.3 Gravimetric and Volumetric Wear
3.4 Cell Responses
4 Notes
References
Chapter 38: Methods for Quantifying Neutrophil Extracellular Traps on Biomaterials
1 Introduction
2 Materials
2.1 Electrospinning
2.2 Cell Culture
2.3 Fluorescent Image Analysis
2.4 NETs-Based ELISA
3 Methods
3.1 Electrospinning
3.2 Fluorescent Image Analysis
3.3 NETs-Based ELISA
4 Notes
References
Chapter 39: In Vivo Imaging of Implanted Hyaluronic Acid Hydrogel Biodegradation
1 Introduction
2 Materials
2.1 Hydrogel Preparation and Optimization of Rheological Properties
2.2 Conjugation of Hydrogels with NIR dyes and Serial Imaging of Hydrogel Degradation
2.3 CEST MRI of Hydrogel Phantoms and Serial Imaging of Hydrogel Degradation
2.4 Stereotactic Injection of Composite Hydrogel in Mice Brain
2.5 In Vivo NIR Imaging
2.6 In Vivo T2-Weighted MRI and CEST MRI
3 Methods
3.1 Hydrogel Preparation and Measurement of Rheological Properties
3.2 Conjugation of Hydrogel Phantoms with NIR Dyes for Two-Color LICOR Imaging
3.3 CEST MRI of Hydrogel Phantoms
3.4 Stereotaxic Implantation of Composite Hydrogels in Mice Brain Striatum
3.5 In Vivo NIR Imaging
3.6 In Vivo T2-Weighted and CEST MRI
4 Notes
References
Chapter 40: Computational Modeling and Simulation to Quantify the Effects of Obstructions on the Performance of Ventricular Ca...
1 Introduction
2 Materials
3 Methods
3.1 Step 1: Identify Input Parameters for Sensitivity Analysis
3.1.1 Step 1a: Define the Catheter Geometry
3.1.2 Step 1b: Specify Other Types of Input Parameters
3.2 Step 2: Model the Ventricle and Implanted Catheter
3.2.1 Step 2a: Create the Mesh
3.2.2 Step 2b: Define Solver Reference Conditions
3.2.3 Step 2c: Specify Boundary Conditions
3.2.4 Step 2d: Validate the Results Through Representative Analysis
3.3 Step 3: Generate Input Files
3.4 Step 4: Perform Analysis on a High Performance Computing (HPC) System
3.5 Step 5: Postprocess the Results
3.6 Step 6: Perform Sensitivity Analysis to Identify the most Influential Parameters
3.7 Step 7: Assign Probability Distributions to the Influential Parameters
3.8 Step 8: Perform Uncertainty Quantification
4 Notes
References
Chapter 41: Selection of Cancer Stem Cell-Targeting Agents Using Bacteriophage Display
1 Introduction
2 Materials
2.1 Transfection/Isolation of GFP+/Chemoresistant CSCs
2.2 Stem Cell Properties
2.2.1 Spheroids
2.2.2 Flow Cytometry
2.2.3 In Vitro Chemoresistance and MTT Assay
2.3 Phage Display
2.3.1 In Vivo Phage Display
2.3.2 Phage Display Selection on Purified Antigen
2.3.3 Phage Display Selection on CSC Line
2.4 Characterizing Phage Using ELISA
2.5 Characterizing Peptides
2.5.1 Peptide ELISA
2.5.2 Live Cell Imaging
3 Methods
3.1 Transfection/Isolation of GFP+/Chemoresistant CSCs
3.2 Confirmation of Stem Cell Properties
3.2.1 Spheroid Formation
3.2.2 Flow Cytometry to Determine Presence of CSC Biomarkers
3.2.3 In Vitro Chemoresistance and MTT Assay
3.3 Phage Display
3.3.1 In Vivo Phage Display Selection
3.3.2 Phage Display Selection Against Purified Antigen
3.3.3 Phage Display Selection on Cancer Stem Cell Line
3.4 Characterizing Phage Using ELISA
3.5 Characterization of Peptides
3.5.1 Peptide ELISA
3.5.2 Live Cell Imaging
4 Notes
References
Chapter 42: Nanoscintillator-Based X-Ray-Induced Photodynamic Therapy
1 Introduction
2 Materials
2.1 Bulk Scintillator Synthesis
2.2 Top Down Nanoparticle Fabrication
2.3 Silica Coating
2.4 Photosensitizer Loading
2.5 Surface Modification
2.6 XEOL Imaging
2.7 X-PDT Administration
3 Methods
3.1 Bulk Scintillator Synthesis
3.2 Top Down Nanoparticle Fabrication
3.3 Silica Coating
3.4 Photosensitizer Loading
3.5 Surface Modification
3.6 XEOL Imaging
3.7 X-PDT Administration
4 Notes
References
Chapter 43: Methods to Measure the Inhibition of ABCG2 Transporter and Ferrochelatase Activity to Enhance Aminolevulinic Acid-...
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Instruments
3 Methods
3.1 Inhibition of ABCG2 Transporter to Enhance ALA-PpIX Fluorescence
3.1.1 Assessment of ABCG2 Transporter Activity in Tumor Cell Lines
3.1.2 Evaluation of Chemicals for the Enhancement of ALA-PpIX
3.2 Inhibition of FECH to Enhance ALA-PpIX Fluorescence
3.2.1 In Vitro FECH Activity Assay
3.2.2 Evaluation of Chemicals on FECH Activity in Cells
3.2.3 Evaluation of Chemicals for the Enhancement of ALA-PpIX
4 Notes
References
Chapter 44: Macroscopic Fluorescence Lifetime Imaging for Monitoring of Drug-Target Engagement
1 Introduction
2 Materials
2.1 Optical Components for ICCD-Gated MFLI
2.2 Optical Components for Hyperspectral MFLI
2.3 Working Components/Optical Filters
2.4 NIR FRET Pairs
2.5 Fluorescently Labeled Antibody and Ligand Probes
2.6 Breast Tumor Xenograft Models
2.7 Immunohistochemical Validation
3 Methods
3.1 Biological Models and Sample Preparation
3.1.1 Probe Labeling and Purification
3.1.2 Breast Tumor Xenograft Models
3.1.3 Tf-TfR In Vivo Imaging Assay
3.1.4 TZM-HER2 In Vivo Imaging Assay
3.1.5 Mice Injections for In Vivo Imaging
3.1.6 Anesthesia and Temperature Control for In Vivo Imaging
3.2 Gated-ICCD MFLI of Biological Models
3.2.1 Setup for Gated-ICCD MFLI Imaging
3.2.2 Data Acquisition Procedure
3.2.3 Intensity Image Retrieval
3.2.4 Lifetime Image Retrieval
3.3 Hyperspectral MFLI of Biological Model
3.3.1 Setup for HMFLI Imaging
3.3.2 Data Acquisition Procedure
3.3.3 Intensity Image Retrieval
3.3.4 Lifetime Image Retrieval
3.4 Immunohistochemical (IHC) Validation
4 Notes
References
Chapter 45: A Method of Tumor In Vivo Imaging with a New Peptide-Based Fluorescent Probe
1 Introduction
2 Materials
2.1 NIR Fluorescence Detection System: Odyssey CLx (LI-COR, Nebraska)
2.2 Cell-Penetrating Peptide
3 Methods
3.1 p28 Synthesis
3.2 NIR Imaging Agent
3.3 Xenograft Tumor Models
3.4 NIR Imaging with the LI-COR Odyssey Imaging System
3.5 Real-Time NIR Imaging In Vivo
3.6 Real-Time NIR Monitoring System, PDE Neo (Mitaka USA Inc., Hamamatsu Photonics)
3.7 Tumor Resection Procedure
4 Notes
References
Chapter 46: Thermal Ablation Treatment for Cervical Precancer (Cervical Intraepithelial Neoplasia Grade 2 or Higher [CIN2+])
1 Introduction
2 Materials
2.1 For all Devices
2.2 For WiSAP Console (See Fig. 1)
2.3 For WiSAP Handheld Device (See Fig. 2)
2.4 Liger Handheld Device (See Fig. 3)
3 Methods
3.1 Preparing the Patient for Treatment (all Devices)
3.2 Treatment with WiSAP Console Thermal Ablator
3.2.1 Preparing the WiSAP Console (before Seeing Patient)
3.2.2 Treatment Application with the WiSAP Console
3.3 Treatment with the WiSAP Handheld Thermal Ablator
3.3.1 Preparing the WiSAP Handheld Device (Before Seeing Patient)
3.3.2 Treatment Application with the WiSAP Handheld Device
3.4 Treatment with the Liger Handheld Thermal Ablator
3.4.1 Preparing the Liger handheld Device (Before Seeing Patient)
3.4.2 Treatment application with the Liger Handheld Device
4 Notes
References
Chapter 47: Employing Novel Porcine Models of Subcutaneous Pancreatic Cancer to Evaluate Oncological Therapies
1 Introduction
2 Materials
2.1 Generating RAG2/IL2RG Deficient Pigs
2.2 Maintaining Cells in Culture
2.3 Harvesting Cells for Injection
2.4 Sterile Passing in of the Cells
2.5 Injecting Tumors
2.6 Monitoring Tumors and Health
2.7 Ultrasound Imaging (See Note 6)
2.8 Necropsy
3 Methods
3.1 Generating RAG2/IL2RG-Deficient Pigs
3.2 Maintaining Cells in Culture
3.3 Harvesting Cells for Injection
3.4 Sterile Passing in of the Cells
3.5 Injecting Tumors
3.6 Monitoring Tumors and Health
3.7 Ultrasound Imaging
3.8 Necropsy
4 Notes
References
Index