This document summarizes methods for using inorganic nanoparticles to probe DNA structure. CdS quantum dots are synthesized through reactions with cadmium nitrate and sodium sulfide. The nanoparticles are then "activated" through addition of cadmium ions, which coats them in a loose web of cadmium. Oligonucleotides of interest are purified by HPLC and annealed to form DNA structures. Titrations of the activated quantum dots with different DNA structures allow inference of local DNA dynamics based on changes in nanoparticle fluorescence.
Nanostructure DNA Templates
Synthesis and Purification of Plasmid templates # Fabrication and Preparation of ultrathin carbon-coated TEM Grids # Preparation of Q-CdS/pUCLeu4 or Q-CdS/φχ174 RF II plasmd samples # their characterization
Quantum-confined cadmium sulfide nanoparticles (Q-CdS) formed circular DNA plasmid pUCLeu4 and φχ174 RF II Quantum confined cadmium sulfide
Surface-Functionalized Nanoparticles for Controlled Drug Delivery: The effectiveness of the surface-functionalized nanoparticles, which consist of copolymers with functional molecules
This document provides an introduction to nanobiotechnology, including its concepts, scope, applications, and future prospects. It defines nanobiotechnology as the combination of nanotechnology and biotechnology, manipulating matter at the nanoscale (1-100 nm) for biological applications. Examples of current applications include growing whole organs like bladders using stem cells, developing targeted cancer drug delivery, and creating polymers to detect metabolites. The future scope may include using molecular manufacturing to program nanobots for delicate surgeries and environmental repair like reconstructing the ozone layer. Overall, the document outlines how nanobiotechnology interfaces biology and nanoscale engineering.
introduction to Nanobiotechnology
what is nanotechnology
bionanotechnology
classical biotechnology industrial production using biological system
modern biotechnology from industrial processes to noval therapeutics
modern biotechnology immunological enzymatic and neucleic acid based technology
Dna based technology
self assembly and supramolecular chemistry
formation of ordered structure at nano scale
DNA Nanotechnology: Concept and its Applications
DNA Nanotechnology # Various 2 and 3 dimensional shapes of DNA nanotechnology # DNA Origami # with their application and Future scope
This document summarizes research on using nanotechnology techniques like thermal induced phase separation (TIPS) to create 3D nanofibrous gelatin scaffolds for tissue engineering applications. The TIPS technique involves dissolving gelatin in a solvent mixture, inducing phase separation at low temperatures, then solvent exchange and freeze-drying to form nanofibrous gelatin matrices. Macroporous gelatin scaffolds were also fabricated by combining TIPS with a porogen leaching method using paraffin spheres. These nanofibrous gelatin scaffolds show potential for tissue engineering due to their ability to mimic the extracellular matrix.
This document summarizes methods for using inorganic nanoparticles to probe DNA structure. CdS quantum dots are synthesized through reactions with cadmium nitrate and sodium sulfide. The nanoparticles are then "activated" through addition of cadmium ions, which coats them in a loose web of cadmium. Oligonucleotides of interest are purified by HPLC and annealed to form DNA structures. Titrations of the activated quantum dots with different DNA structures allow inference of local DNA dynamics based on changes in nanoparticle fluorescence.
Nanostructure DNA Templates
Synthesis and Purification of Plasmid templates # Fabrication and Preparation of ultrathin carbon-coated TEM Grids # Preparation of Q-CdS/pUCLeu4 or Q-CdS/φχ174 RF II plasmd samples # their characterization
Quantum-confined cadmium sulfide nanoparticles (Q-CdS) formed circular DNA plasmid pUCLeu4 and φχ174 RF II Quantum confined cadmium sulfide
Surface-Functionalized Nanoparticles for Controlled Drug Delivery: The effectiveness of the surface-functionalized nanoparticles, which consist of copolymers with functional molecules
This document provides an introduction to nanobiotechnology, including its concepts, scope, applications, and future prospects. It defines nanobiotechnology as the combination of nanotechnology and biotechnology, manipulating matter at the nanoscale (1-100 nm) for biological applications. Examples of current applications include growing whole organs like bladders using stem cells, developing targeted cancer drug delivery, and creating polymers to detect metabolites. The future scope may include using molecular manufacturing to program nanobots for delicate surgeries and environmental repair like reconstructing the ozone layer. Overall, the document outlines how nanobiotechnology interfaces biology and nanoscale engineering.
introduction to Nanobiotechnology
what is nanotechnology
bionanotechnology
classical biotechnology industrial production using biological system
modern biotechnology from industrial processes to noval therapeutics
modern biotechnology immunological enzymatic and neucleic acid based technology
Dna based technology
self assembly and supramolecular chemistry
formation of ordered structure at nano scale
DNA Nanotechnology: Concept and its Applications
DNA Nanotechnology # Various 2 and 3 dimensional shapes of DNA nanotechnology # DNA Origami # with their application and Future scope
This document summarizes research on using nanotechnology techniques like thermal induced phase separation (TIPS) to create 3D nanofibrous gelatin scaffolds for tissue engineering applications. The TIPS technique involves dissolving gelatin in a solvent mixture, inducing phase separation at low temperatures, then solvent exchange and freeze-drying to form nanofibrous gelatin matrices. Macroporous gelatin scaffolds were also fabricated by combining TIPS with a porogen leaching method using paraffin spheres. These nanofibrous gelatin scaffolds show potential for tissue engineering due to their ability to mimic the extracellular matrix.
Protein based nanostructures for biomedical applications karoline Enoch
Proteins are kind of natural molecules that show unique
functionalities and properties in biological materials and
manufacturing feld. Tere are numerous nanomaterials
which are derived from protein, albumin, and gelatin. Tese
nanoparticles have promising properties like biodegradability, nonantigenicity, metabolizable, surface modifer, greater
stability during in vivo during storage, and being relatively
easy to prepare and monitor the size of the particles.
These particles have the ability to attach covalently with
drug and ligand
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Richard Feynman is credited with the birth of nanotechnology in 1959 when he challenged scientists that manipulating matter at the nanoscale was possible if the laws of physics allowed. Nanobiotechnology was initiated in 1980 with the development of atomic force microscopy that enables atomic-level imaging. Nanobiotechnology involves creating functional materials and devices through understanding and controlling matter at the nanometer scale of 1 to 100 nm, where new properties emerge. Applications include biomedical imaging, advanced drug delivery, biosensing, and regenerative medicine.
Nanobiotechnology BY Neelima Sharma,WCC CHENNAI,neelima.sharma60@gmail.comNeelima Sharma
Nanobiotechnology combines nanotechnology and biotechnology to create functional materials and devices at the nanoscale (1-100 nm) where new properties emerge. It allows for imaging and manipulation of biomolecules, development of biosensors, targeted drug delivery for cancer treatment, and regenerative medicine using biomimetic tissues. While offering promising applications, nanobiotechnology may also pose toxicity risks if nanoparticles are able to penetrate tissues and cells and cause biochemical damage.
Applications of nanobiotechnology by kk sahuKAUSHAL SAHU
INTRODUCTION
DEFINITION
HISTORY
NANOSCALE
NANOPARTICLES
NANOBIOTECHNOLOGY
NANOTOOLS
APPLICATIONS
RESEARCH
CONCLUSION
REFRENCES
Nanotechnology is the design, characterization and application of structures, devices and systems by controlling shape and size at the nanometer scale!” defines the Royal Academy of Engineering in London in 2004 .
Concepts that are enhanced through nanobiology include: nanodevices, nanoparticles, and nanoscale phenomena that occurs within the discipline of nanotechnology.
Introduction
Definition
History
Advantages of nanobiotechnology
Applications of nanobiotechnology
Drawback of nanobiotechnology
New features in the nanobiotechnology
Conclusion
References
The document discusses various applications of nanotechnology in biomedical fields such as medicine and healthcare. It describes how nanotechnology can be used to develop targeted drug delivery systems, lab-on-chip devices for disease detection and diagnosis, bionanomaterials for medical applications, and nanoscale machines and sensors. It also discusses how nanotechnology enables more precise detection and treatment of diseases like cancer at the molecular level with fewer side effects.
Cancer is caused by changes in gene expression or mutations that lead to abnormal cell growth. The presentation discusses the types and properties of cancer cells, causes like carcinogenic agents and viruses, tumor suppressor genes like p53 and oncogenes. Proteomics is the study of the complete set of proteins in a cell or organism and techniques used include biomarkers, 2D gel electrophoresis, and mass spectrometry like MALDI-TOF and SELDI-TOF. Improvements in multidimensional separations and nanotechnology may help identify more biomarkers and develop novel cancer diagnostics and therapeutics.
This document discusses DNA-based nanobioelectronics. It begins by introducing DNA and its potential use in nanoelectronics. It then discusses using DNA to template the assembly of metal nanoparticles, as well as using sequence-specific molecular lithography to pattern nanoparticles on DNA. It also discusses using metal nanoparticles for DNA detection and field-effect transistors for label-free electronic DNA detection. The document outlines applications in areas like medical diagnostics, environmental monitoring, and more. It concludes by discussing using DNA derivatives that may have better intrinsic conductivity than DNA.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
PPT on "Functionalization of Nanoparticles and Nanoplatelets" by Deepak rawalDeepak Rawal
Presentation on Functionalization of nanoparticles, magnetic nanoparticles, chemical funtionalization, funtionalization of carbon nanotubes and their applications. Introduction about graphite nanoplatelets.
This document provides an overview of DNA microarrays (DNA chips). It discusses that DNA chips allow scientists to simultaneously measure gene expression levels or genotype multiple genomic regions. It describes the principle technologies used in DNA chips, including attaching cDNA or oligonucleotide probes to glass or silicon surfaces. The document also provides background on DNA and microarrays, their history, applications in gene expression analysis and disease research, and principle of hybridization. It discusses alternative bead-based array technologies and how microarrays enabled large-scale genomic experiments.
Bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment. Bioreactors have been used in animal cell culture since the 1980s in order to produce vaccines and other drugs and to culture large cell populations. Bioreactors for use in tissue engineering have progressed from such devices.
A tissue engineering bioreactor can be defined as a device that uses mechanical means to influence biological processes. In tissue engineering, this generally means that bioreactors are used to stimulate cells and encourage them to produce extracellular matrix (ECM). There are numerous types of bioreactor which can be classified by the means they use to stimulate cells.
This document summarizes self-assembly of DNA structures. It discusses how DNA can be used as a nanoscale building material through sticky ends, branches, and double crossover structures that allow strands to selectively bind. DNA tiles and origami are introduced, where tiles and helper strands are used to form two-dimensional crystalline assemblies and predefined shapes from a long scaffold strand. The ability of DNA to self-assemble through complementary base pairing allows for precise nanostructures to be designed and fabricated from DNA alone.
This document discusses nanotechnology and its applications in medicine. It begins with the origins and definitions of nanotechnology. Some key approaches to nanofabrication include top-down and bottom-up methods. Nanocarriers such as liposomes, dendrimers, micelles, and nanoparticles can be used for targeted drug delivery. Nanotechnology has applications in regenerative medicine, disease diagnosis using nanomolecular diagnostics, and in-vitro diagnostics including nano biosensors and nanoarrays. Overall, nanomedicine holds promise for earlier disease detection and more targeted treatment approaches.
Nanomedicines show promise for improving cancer treatment. Nanoparticles can be engineered to target cancer cells specifically and deliver toxic payloads or heat. Gold nanoparticles activated by laser light can hyperthermically destroy tumor cells from the inside. Challenges remain in developing nanoparticles that are safe and can effectively reach tumors. If these challenges can be addressed, nanomedicine may enable more precise cancer detection and treatments with fewer side effects than conventional therapies.
Protein-protein interactions (PPIs) are important for many cellular functions. There are two main types of PPIs - transient interactions which are brief, and stable interactions which form multiprotein complexes. Crosslinking can capture both transient and stable PPIs by covalently binding interacting proteins. In vivo crosslinking studies PPIs in their native environment while in vitro crosslinking allows better reaction control. Pull-down assays use affinity purification to isolate stable protein complexes and identify binding partners of a bait protein. SDS-PAGE is commonly used to separate and visualize proteins isolated by techniques like pull-down.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
Nanotechnology involves manipulating matter at the atomic or molecular scale. It allows scientists to build structures to specific atomic specifications ranging from 1 to 100 nanometers in size. Key tools used in nanotechnology include scanning probes like atomic force microscopes and techniques like lithography which can precisely construct nanostructures. Potential applications of nanotechnology include more effective drug delivery systems, stronger and lighter materials, flexible electronics, and advanced computer chips. While nanotechnology promises many benefits, potential risks also exist and more research is still needed to realize its future possibilities while avoiding unintended consequences.
Nanotechnology involves manipulating matter at the atomic and molecular scale. It has many applications in fields like electronics, materials science, medicine, and more. Some key points:
- It allows engineering of functional systems at the nanometer scale (1-100 nm) which is around the size of atoms and molecules.
- Tools like atomic force microscopes and scanning tunneling microscopes enabled the study and engineering of matter at the nanoscale.
- Nanotechnology is used in areas like drug delivery, cancer treatment, stain-resistant and antibacterial fabrics, flexible electronics, solar cells, and more powerful computers.
- India has initiatives like the Nano Science and Technology Initiative and Nanoscience and Technology Mission
Protein based nanostructures for biomedical applications karoline Enoch
Proteins are kind of natural molecules that show unique
functionalities and properties in biological materials and
manufacturing feld. Tere are numerous nanomaterials
which are derived from protein, albumin, and gelatin. Tese
nanoparticles have promising properties like biodegradability, nonantigenicity, metabolizable, surface modifer, greater
stability during in vivo during storage, and being relatively
easy to prepare and monitor the size of the particles.
These particles have the ability to attach covalently with
drug and ligand
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Richard Feynman is credited with the birth of nanotechnology in 1959 when he challenged scientists that manipulating matter at the nanoscale was possible if the laws of physics allowed. Nanobiotechnology was initiated in 1980 with the development of atomic force microscopy that enables atomic-level imaging. Nanobiotechnology involves creating functional materials and devices through understanding and controlling matter at the nanometer scale of 1 to 100 nm, where new properties emerge. Applications include biomedical imaging, advanced drug delivery, biosensing, and regenerative medicine.
Nanobiotechnology BY Neelima Sharma,WCC CHENNAI,neelima.sharma60@gmail.comNeelima Sharma
Nanobiotechnology combines nanotechnology and biotechnology to create functional materials and devices at the nanoscale (1-100 nm) where new properties emerge. It allows for imaging and manipulation of biomolecules, development of biosensors, targeted drug delivery for cancer treatment, and regenerative medicine using biomimetic tissues. While offering promising applications, nanobiotechnology may also pose toxicity risks if nanoparticles are able to penetrate tissues and cells and cause biochemical damage.
Applications of nanobiotechnology by kk sahuKAUSHAL SAHU
INTRODUCTION
DEFINITION
HISTORY
NANOSCALE
NANOPARTICLES
NANOBIOTECHNOLOGY
NANOTOOLS
APPLICATIONS
RESEARCH
CONCLUSION
REFRENCES
Nanotechnology is the design, characterization and application of structures, devices and systems by controlling shape and size at the nanometer scale!” defines the Royal Academy of Engineering in London in 2004 .
Concepts that are enhanced through nanobiology include: nanodevices, nanoparticles, and nanoscale phenomena that occurs within the discipline of nanotechnology.
Introduction
Definition
History
Advantages of nanobiotechnology
Applications of nanobiotechnology
Drawback of nanobiotechnology
New features in the nanobiotechnology
Conclusion
References
The document discusses various applications of nanotechnology in biomedical fields such as medicine and healthcare. It describes how nanotechnology can be used to develop targeted drug delivery systems, lab-on-chip devices for disease detection and diagnosis, bionanomaterials for medical applications, and nanoscale machines and sensors. It also discusses how nanotechnology enables more precise detection and treatment of diseases like cancer at the molecular level with fewer side effects.
Cancer is caused by changes in gene expression or mutations that lead to abnormal cell growth. The presentation discusses the types and properties of cancer cells, causes like carcinogenic agents and viruses, tumor suppressor genes like p53 and oncogenes. Proteomics is the study of the complete set of proteins in a cell or organism and techniques used include biomarkers, 2D gel electrophoresis, and mass spectrometry like MALDI-TOF and SELDI-TOF. Improvements in multidimensional separations and nanotechnology may help identify more biomarkers and develop novel cancer diagnostics and therapeutics.
This document discusses DNA-based nanobioelectronics. It begins by introducing DNA and its potential use in nanoelectronics. It then discusses using DNA to template the assembly of metal nanoparticles, as well as using sequence-specific molecular lithography to pattern nanoparticles on DNA. It also discusses using metal nanoparticles for DNA detection and field-effect transistors for label-free electronic DNA detection. The document outlines applications in areas like medical diagnostics, environmental monitoring, and more. It concludes by discussing using DNA derivatives that may have better intrinsic conductivity than DNA.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
PPT on "Functionalization of Nanoparticles and Nanoplatelets" by Deepak rawalDeepak Rawal
Presentation on Functionalization of nanoparticles, magnetic nanoparticles, chemical funtionalization, funtionalization of carbon nanotubes and their applications. Introduction about graphite nanoplatelets.
This document provides an overview of DNA microarrays (DNA chips). It discusses that DNA chips allow scientists to simultaneously measure gene expression levels or genotype multiple genomic regions. It describes the principle technologies used in DNA chips, including attaching cDNA or oligonucleotide probes to glass or silicon surfaces. The document also provides background on DNA and microarrays, their history, applications in gene expression analysis and disease research, and principle of hybridization. It discusses alternative bead-based array technologies and how microarrays enabled large-scale genomic experiments.
Bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment. Bioreactors have been used in animal cell culture since the 1980s in order to produce vaccines and other drugs and to culture large cell populations. Bioreactors for use in tissue engineering have progressed from such devices.
A tissue engineering bioreactor can be defined as a device that uses mechanical means to influence biological processes. In tissue engineering, this generally means that bioreactors are used to stimulate cells and encourage them to produce extracellular matrix (ECM). There are numerous types of bioreactor which can be classified by the means they use to stimulate cells.
This document summarizes self-assembly of DNA structures. It discusses how DNA can be used as a nanoscale building material through sticky ends, branches, and double crossover structures that allow strands to selectively bind. DNA tiles and origami are introduced, where tiles and helper strands are used to form two-dimensional crystalline assemblies and predefined shapes from a long scaffold strand. The ability of DNA to self-assemble through complementary base pairing allows for precise nanostructures to be designed and fabricated from DNA alone.
This document discusses nanotechnology and its applications in medicine. It begins with the origins and definitions of nanotechnology. Some key approaches to nanofabrication include top-down and bottom-up methods. Nanocarriers such as liposomes, dendrimers, micelles, and nanoparticles can be used for targeted drug delivery. Nanotechnology has applications in regenerative medicine, disease diagnosis using nanomolecular diagnostics, and in-vitro diagnostics including nano biosensors and nanoarrays. Overall, nanomedicine holds promise for earlier disease detection and more targeted treatment approaches.
Nanomedicines show promise for improving cancer treatment. Nanoparticles can be engineered to target cancer cells specifically and deliver toxic payloads or heat. Gold nanoparticles activated by laser light can hyperthermically destroy tumor cells from the inside. Challenges remain in developing nanoparticles that are safe and can effectively reach tumors. If these challenges can be addressed, nanomedicine may enable more precise cancer detection and treatments with fewer side effects than conventional therapies.
Protein-protein interactions (PPIs) are important for many cellular functions. There are two main types of PPIs - transient interactions which are brief, and stable interactions which form multiprotein complexes. Crosslinking can capture both transient and stable PPIs by covalently binding interacting proteins. In vivo crosslinking studies PPIs in their native environment while in vitro crosslinking allows better reaction control. Pull-down assays use affinity purification to isolate stable protein complexes and identify binding partners of a bait protein. SDS-PAGE is commonly used to separate and visualize proteins isolated by techniques like pull-down.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
Nanotechnology involves manipulating matter at the atomic or molecular scale. It allows scientists to build structures to specific atomic specifications ranging from 1 to 100 nanometers in size. Key tools used in nanotechnology include scanning probes like atomic force microscopes and techniques like lithography which can precisely construct nanostructures. Potential applications of nanotechnology include more effective drug delivery systems, stronger and lighter materials, flexible electronics, and advanced computer chips. While nanotechnology promises many benefits, potential risks also exist and more research is still needed to realize its future possibilities while avoiding unintended consequences.
Nanotechnology involves manipulating matter at the atomic and molecular scale. It has many applications in fields like electronics, materials science, medicine, and more. Some key points:
- It allows engineering of functional systems at the nanometer scale (1-100 nm) which is around the size of atoms and molecules.
- Tools like atomic force microscopes and scanning tunneling microscopes enabled the study and engineering of matter at the nanoscale.
- Nanotechnology is used in areas like drug delivery, cancer treatment, stain-resistant and antibacterial fabrics, flexible electronics, solar cells, and more powerful computers.
- India has initiatives like the Nano Science and Technology Initiative and Nanoscience and Technology Mission
Nanotechnology involves manipulating matter at the atomic and molecular scale. It has various applications in fields like electronics, materials, medicine and more. Some key points:
1. It allows developing new materials and devices with improved properties by controlling structures at the nanoscale.
2. Tools like atomic force microscopes and scanning tunneling microscopes enabled research. Carbon nanotubes, nanorods and nanobots are examples of nanomaterials.
3. Applications include using silver nanoparticles and carbon nanotubes in fabrics and medicines, developing flexible electronics and improving computer chips.
Nanotechnology involves manipulating matter at the atomic or molecular scale. It has many potential applications in areas like medicine, electronics, materials and computing. Some key points:
- It allows precise engineering at the nanoscale of 1-100 nanometers. Tools like STMs and AFMs are used.
- Applications include carbon nanotubes for strong lightweight materials, quantum dots for displays, and nanobots potentially for drug delivery and environmental remediation.
- Challenges include potential health effects of nanoparticles and risks of military applications like self-replicating viruses or runaway nanobots. Both top-down and bottom-up assembly approaches are used in nanotechnology.
Nanotechnology allows the precise placement of small structures at low cost, leading to economic growth, enhanced security, improved quality of life, and job creation. There are top-down and bottom-up approaches to nanoscale fabrication. Key tools include carbon nanotubes, quantum dots, and nanobots. Carbon nanotubes have exceptional strength and can penetrate cell walls, making them useful for applications like cancer treatment, sensors, electronics, and solar cells. Quantum dots can be used in displays and MEMS due to their reflectivity properties. Nanobots only a few nanometers in size could count molecules and potentially be used for detection, drug delivery, and biomedical instrumentation. Nanotechnology has many applications including electronics, energy,
This document provides an overview of nanotechnology. It defines nanotechnology as the study and engineering of matter at the nanoscale, or atomic level. The document outlines the history of nanotechnology from its conception in 1959 to modern applications. Key tools used in nanotechnology like atomic force microscopes and carbon nanotubes are described. The document also discusses different approaches (top-down vs bottom-up), materials used, and applications of nanotechnology in areas like drugs, fabrics, electronics, and computers. It provides examples of how nanotechnology is enhancing performance in these domains.
This presentation is a simple explain of Nano-springs which introduce this Nano-materials easily. You can use this PPTx File to present in your class and seminars as well. We prepare this file to present in Tabriz University of Medical Sciences when We were MSc Medical Nanotechnology student. It will be useful for you too.
Nanotechnology is the manipulation of matter on an atomic, molecular, and supramolecular scale. The three key points are:
1) The concept of nanotechnology was first proposed in 1959, but it emerged in the 1980s with advances like the scanning tunneling microscope and discovery of fullerenes. The term was coined in 1974.
2) Nanotechnology involves engineering materials and devices within the size range of 1-100 nanometers. At this scale, the properties of materials differ from those at larger scales.
3) Potential applications of nanotechnology include electronics, energy storage, drug delivery, biotechnology, and new materials with unique properties. It is estimated nanotechnology will become a trillion dollar market by
This document discusses the topic of nanotechnology and its applications. It begins with an overview of nanotechnology, defining it as the manipulation of materials at the nanoscale (less than 100 nanometers). It then describes the two main approaches to nanotechnology - top-down and bottom-up. Several types of nanomaterials are discussed, including carbon nanotubes, graphene, fullerenes. The document concludes by outlining several applications of nanotechnology, such as in sensors, medicine, environmental remediation, food science, and electronics.
This document provides an overview of nanotechnology. It begins with definitions of nanotechnology as the study and manipulation of matter at the atomic scale, with a nanometer being one billionth of a meter. The document then discusses the history of nanotechnology from Richard Feynman's 1959 talk introducing the concept to modern developments like the scanning tunneling microscope. Tools and techniques used in nanotechnology like lithography and microscopes are described. Specific nanomaterials like carbon nanotubes, nanorods, and nanobots are explained. The wide applications of nanotechnology in areas like electronics, medicine, fabrics and more are outlined. The future potential of nanotechnology is also mentioned.
Nanotechnology involves building devices at the molecular scale, typically less than 100 nanometers. It has applications in fields like computer science, medicine, robotics, and electronics. In medicine, nanorobots could help deliver drugs, monitor health, and even transport oxygen in the bloodstream. Researchers are also exploring using nanotechnology to build smaller computer chips, develop molecular machines like nanomotors, and create nanotubes for applications such as ultra-strong bearings.
This document discusses nanomedicine and various nanoscale structures that can be used for medical applications. It begins by explaining how nanotechnology allows analysis and repair of the human body at the molecular level. It then describes various nanoscale structures like liposomes, dendrimers, carbon nanotubes, quantum dots, mesoporous silica nanoparticles and their properties. These nanoparticles can be used for targeted drug delivery, imaging and diagnosis. The document also discusses some current and potential applications of these nanotechnologies in areas like cancer treatment, biomolecular sensing and gene therapy.
This document discusses carbon nanotubes (CNTs). It defines nanochemistry as the study and synthesis of nanoparticles between 1-100 nanometers in size. CNTs are cylindrical carbon molecules with unusual properties valuable for applications in nanotechnology, electronics, optics, and other fields. There are two main types of CNTs - single-walled and multi-walled. CNTs can be produced through methods like arc discharge, laser ablation, and chemical vapor deposition. CNTs have remarkable mechanical, electrical, and thermal properties and are being researched for applications in areas like medicine, composites, microelectronics, chemicals, and more, though more study is still needed on their toxicity and environmental impact.
CARBON NANO TUBE -- PREPARATION – METHODSArjun K Gopi
The document discusses carbon nanotubes, including their structure and properties. It describes three common production methods: arc discharge, laser ablation, and chemical vapor deposition. Arc discharge was the initial discovery method and remains widely used, but it produces impurities. Laser ablation yields primarily single-walled nanotubes but is expensive. Chemical vapor deposition allows control over diameter and is suitable for scaling up. Purification techniques are needed to separate nanotubes from byproducts. Potential applications include electronics, energy storage, and reinforced composites.
This document provides an introduction to nanotechnology, including key concepts and applications. It discusses how nanotechnology works at the atomic scale using techniques like scanning probe microscopes. Examples of nanoparticles and their uses in areas like drug delivery, disease detection, and imaging are provided. Both current applications and future potential are explored, with medical applications being a major focus. Some concerns about potential negative biological effects of nanoparticles are also noted.
Construction and design of a novel drug delivery systemBalaganesh Kuruba
This document discusses using gold nanospheres for drug and gene delivery. It begins by introducing nanotechnology and some of its applications in fields like bioremediation. It then discusses problems with existing drug delivery systems like solubility issues and cytotoxicity. It describes how gold nanospheres will be fabricated using a sacrificial galvanic replacement method and coated with molecules like epsin to enter cells. A genetic construct containing marker and therapeutic genes will be encapsulated. The spheres will be detected using surface enhanced Raman scattering detected by an optical probe. Overall, the document proposes using gold nanospheres as a new drug and gene delivery system to address issues with current technologies.
Nanotechnology involves manipulating matter at the atomic scale to build functional systems. It allows engineering at the micro and atomic levels. A nanometer is one billionth of a meter. The concept was first proposed in 1959 and scanning tunneling microscopes and buckyballs in the 1980s helped establish the field. Carbon nanotubes have an extremely high length-to-diameter ratio and unique mechanical and electrical properties. They are being used in applications like lightweight bicycles, boats, and electronics. Nanoparticles exhibit surface plasmon resonance that gives color and depends on size and shape. Nanotechnology shows promise in areas like cancer treatment through targeted drug delivery, stain-resistant fabrics, flexible electronics, and future possibilities like nanorobotics and
This document discusses nanomedicine and applications of nanotechnology in medicine. It begins by explaining how nanotechnology allows analysis and repair of the human body at the molecular level. It then covers major biological structures and scales. Richard Feynman's seminal 1959 talk inspiring nanotechnology is mentioned. The document defines nanomedicine and discusses markets, applications including drug delivery and disease detection, nanoparticles, carbon nanotubes, and their properties and potential uses in medicine. In particular, carbon nanotubes are discussed as potential drug delivery vessels that can be functionalized for targeting and controlled release.
Genetic Engineering of Male Sterility for Hybrid Seed Production # Methods of Hybrid Seed Production - Hybridization techniques # Examples of Male Sterile Hybrid Seed
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
Dr. Divya Sharma is an assistant professor at A Biodiction. The document discusses the process of creating recombinant plasmids by inserting a gene or DNA fragment of interest into a circular piece of DNA called a plasmid using restriction enzymes and ligase. This recombinant DNA can then be used to create transgenic plants by modifying tumor-inducing genes (Ti-plasmids) that lack transferable genes and inserting the gene of interest instead. The document then discusses various types of Agrobacterium strains used, the regeneration and selection process for transgenic plants, and methods for detecting and characterizing the inserted trait genes. Some examples of commercial transgenic crops are also provided, such as golden rice engineered for vitamin A production and herb
The document discusses plant protoplast isolation, purification, and culturing. Some key points:
- Protoplasts are plant cells that have had their cell walls removed, leaving just the plasma membrane. They allow for plant cell fusion and regeneration.
- Protoplasts are typically isolated from plant tissues like leaves using enzymatic digestion with cellulase and pectinase. This yields more protoplasts than mechanical methods.
- Isolated protoplasts are purified by centrifugation and washing to remove cell debris. They are then cultured in liquid or solid nutrient media and tested for viability before regeneration.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
1. Somatic embryogenesis is a process where embryos are formed from somatic plant cells, such as leaf cells, that are not normally involved in embryo formation. 2. These somatic embryos develop through similar stages as zygotic embryos, including globular, heart-shaped, torpedo-shaped, and cotyledonary stages. 3. Somatic embryogenesis can occur through direct embryogenesis from explant tissue or indirect embryogenesis which involves first forming a callus that then develops embryos.
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
Micropropagation is a tissue culture technique used for the rapid asexual propagation of plants. It involves culturing small pieces of plant tissues or organs on growth media in controlled conditions. The process includes initiation of cultures from explants, multiplication through shoot proliferation or somatic embryogenesis, rooting of shoots, and transplantation of plantlets to soil. Micropropagation allows for large-scale production of genetically identical clones in a short period of time. It has applications in producing disease-free plants, conserving rare species, and commercializing new plant varieties.
This document summarizes the culture of in-vitro pollination and fertilization. It describes the different types of in-vitro pollination including ovular, ovarian, placental and stigmatic pollination. The methods of in-vitro pollination and fertilization are outlined involving sterilization procedures and suitable media and explants. Applications include using in-vitro fertilization to overcome self-incompatibility in some plants or enable intergeneric crosses. The techniques used involve isolating pollen and egg cells, inducing fusion through electrofusion, and culturing the fertilized eggs on nutrient media to develop into plants. Considerations for successful in-vitro fertilization include the physiological state of
Dr. Divya Sharma discusses plant growth regulators and their roles in plant growth and development. There are internal and external factors that influence plant growth. Hormones called phytohormones regulate many processes including flowering, stem and leaf formation, fruit ripening, and more. The major classes of plant growth regulators are auxins, cytokinins, gibberellins, ethylene, and abscisic acid. Auxins promote cell elongation and root formation while cytokinins stimulate cell division and shoot growth. Gibberellins promote stem elongation and flowering. Together these hormones precisely control plant growth and differentiation.
Quantum dots are nanocrystalline semiconductor particles between 1-10 nm in size that display quantized energy levels. Their spectral properties depend on their size - smaller quantum dots emit higher energy light while larger ones emit lower energy. This allows using quantum dots of different sizes to produce a range of colors. Potential applications of quantum dots include use in TV and display technologies due to their pure colors and long lifetimes. Quantum dots are also being explored for biological and chemical applications such as cancer treatment where they can target organs more precisely than conventional drugs.
plant Biotechnology: The application of Plant Biotechnology by use of scientific method to manipulate living cells or organisms for practical uses (manipulation and transfer of genetic material).
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
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Synthetic Nanoscale Elements for Delivery of Material into Viable Cells
1. Synthetic Nanoscale Elements for
Delivery of Material into Viable Cells
Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)
2. Introduction
■ As they reside at the same size scale as the biomolecular machines of
cells, engineered nanoscale devices may provide the means to
construct new tools for monitoring and manipulating cellular
processes.
■ The ideal tools would interface directly with subcellular,
biomolecular processes, allowing the control of these processes while
also providing transduction of responses with both spatial and
temporal resolution. Ultimately, this interfacing might be performed
without adversely affecting cell viability or functionality.
■ Investigators have highlighted this interface of nanotechnology and
biotechnology through the use of non-bleaching fluorescent
nanocrystals in place of dyes as a means of monitoring cellular
processes.
Timothy E.,McKnght, et al. NanoBiotechnology Protocols, Methods in Molecular Biology; Vol 303
3. ■ Pulled-glass capillaries with nanoscale tips have been implemented for
cellular and subcellular electrophysiological stimulus and monitoring
and for manipulating intracellular material using microinjection of
membrane-impermeable molecules (e.g., proteins, DNA).
■ Conventionally, these devices have required manipulating cells one at
a time while visualizing the process under a microscope and, thus,
provide a serial interface to individual cells.
■ Parallel embodiments of these devices have been fabricated using
silicon microfabrication methods, but as with all micromachining
techniques, there are limitations to the ultimate size scale and density
of features (tip radii and spacing of the silicon needles) and to the
choice of substrate materials (i.e., parallel embodiments not easily
fabricated on transparent substrates convenient for cell culture)
■ By contrast, recent advances in the synthesis of nanomaterials,
including carbon nanofibers and carbon nanotubes, can avoid these
limitations and provide the means to construct massively parallel,
addressable functional nanoscale devices including chemically
specific atomic force microscope probes, electrochemical probes, and
electromechanical manipulators.
4. ■ Deterministic arrays of closely spaced (pitch ≥ 1 µm) vertically
aligned carbon nanofibers (VACNFs) may be grown on a wide
variety of substrates (including quartz and glass slides) with wide
bases that provide mechanical strength while still generating a small
diameter tip (≥5-nm tip radius) appropriate for insertion directly into
cells.
■ Describe the steps for fabricating functional VACNF devices and for
implementing these devices in a first step toward molecular-scale
integration with live cells.
■ As a demonstration, these devices are used to introduce exogenous
material (e.g., nanofiber-scaffolded plasmid DNA) into viable cells.
■ While providing a novel and effective method for gene delivery, the
functional insertion of nanofiber-scaffolded DNA has exciting
potential for ultimately providing a nanoscale genomic interfacing
platform [Note 1]
Timothy E.,McKnght, et al. NanoBiotechnology Protocols, Methods in Molecular Biology; Vol 303
5. Materials Required
Nanofiber Synthesis
■ Photoresist (Shipley)
■ Si wafers (Silicon Quest)
■ Ni slugs (Goodfellows)
■ Acetone and isopropanol
■ Dry nitrogen
■ Acetylene (Matheson gases)
■ Ammonia (Matheson gases)
Nanofiber-mediated
Material Delivery
■ Plasmid DNA: pd2EYFP-N1 or
pgreenlantern-1
■ 100 mM 2-(N-morpholino) ethane
sulfonic acid (MES) buffer
adjusted to pH 4.5–4.8 with NaOH
■ 1-Ethyl-3-(3-dimethyl-amino-
propyl) carbodiimide (EDC)
■ Phosphate-buffered saline (PBS) or
other serum-free and dye-free cell
suspension buffer
■ Eppendrof micro-centrifuge tubes
■ Sylgard 184 two-part poly-
(dimethylsiloxane) (PDMS) kit
■ Propidium iodide
6. Methods
1. Synthesis of VACNFs (Vertically aligned carbon nanofibers)
2. Spotting of Nanofiber Arrays With Plasmid DNA
3. Covalent Modification of Fibers With Plasmid DNA
4. Preparation of Microcentrifuge Spin Tubes
5. Interfacing of Cells onto Carbon Nanofiber Arrays
Timothy E.,McKnght, et al. NanoBiotechnology Protocols, Methods in Molecular Biology; Vol 303
7. Synthesis of VACNFs
(Vertically aligned carbon nanofibers)
■ VACNFs are synthesized deterministically using catalytically
controlled, direct current (DC) plasma-enhanced chemical vapor
deposition.
■ VACNFs grow from catalyst particles (e.g., Ni) deposited onto a
substrate at predefined locations when placed onto a cathode of a
glow discharge system at elevated temperature (e.g., 700°C) in a
flow of acetylene and ammonia.
■ Carbon species decompose at the surface of the catalyst particle, and
free carbon atoms diffuse through it and are incorporated into a
growing nanostructure between the particle and the substrate.
8. Synthesis of
VACNFs by
PECVD process
Schematic representation of
the PECVD process for
growing vertically aligned
carbon nanofibers.
a). Catalyst pretreatment/
nanoparticles formation;
b). Growth of carbon
nanofibers
9. ■ Such nanofibers, which have a catalyst particle at the tip, grow
oriented along the electric field lines. Thus, the orientation can be
determined by the direction of the field.
■ The lateral dimensions of the nanofibers are determined by the size
of the catalytic particle, and their length can be precisely controlled
by the growth time.
■ Growth conditions can be manipulated to control the shape of the
nanofibers (e.g., conical vs cylindrical) and to control the chemical
composition of material deposited on the walls (e.g., amorphous
carbon or silicon nitride).
■ The next specific steps for the synthesis of VACNFs suitable for
functional integration into viable cells. An example of such an array
is shown in Figure.
10. TEM images of
Nanofiber:
Viable cells of
VACNFs
Typical array of VACNFs
suitable for integration with
viable cells (inset)
Transmission electron
microscope (TEM) image of
nanofiber
11. a. Patterning of Substrate
I. Spin a photoresist or an electron beam resist (depending on
whether photolithography or electron beam lithography is used,
respectively) on the surface of a silicon wafer and process
according to the manufacturer’s guidelines.
II. Expose and develop a pattern so that the resist is removed from the
areas where the carbon nanofibers will be grown.
Specific details depend on the lithography tool and resist. For a general
overview of the processes. For transfection of mammalian cells, such as
Chinese hamster ovary (CHO) cells, the pattern is an array of dots that
are 0.5 μm in diameter arranged on a square grid with 5-μm spacing.
Additional grid lines every 100 μm and indexing numbers are helpful to
track transfected cells.
12. b. Deposition of Catalyst
I. Load patterned wafers into a physical vapor deposition chamber,
and after an appropriate vacuum is achieved (10^–7 torr), deposit
20 nm of Ni.
13. c. Lifting Off of Excess Metal
I. Place a metallized wafer in a glass dish filled with acetone, cover
the dish with a looking-glass cover to prevent evaporation of
acetone, and soak it for 30–60 min.
II. Ultrasonicate for 30 s.
III. Remove the wafer from the dish but do not let the acetone dry out,
and while holding the wafer with tweezers above the dish
immediately rinse the wafer with a spray of acetone to remove
metal particulates. Wash the acetone by spraying the wafer with
isopropanol.
IV. Blow-dry with N2.
14. d. Growth of Carbon Nanofibers
Growth parameters such as gas flows, pressure, and plasma current will
vary depending on the particular chamber setup, type of substrate,
chamber and substrate size, and catalyst pattern.
i. Mount a wafer on top of the heater-cathode of a DC plasma
enhanced chemical vapor deposition (PECVD) system.
ii. Evacuate the chamber until a vacuum below 0.1 torr is achieved.
iii. Set the substrate temperature controller to 700°C.
iv. Introduce ammonia by opening the automatic valve and set the flow
to 80 standard cubic centimeters per minute (sscm) and pressure to
3 torr.
v. When the temperature reaches 700°C, introduce acetylene at 20
sccm.
15. vi. Ten seconds after opening the acetylene valve, start DC plasma
discharge by turning on the high voltage. Set the high-voltage
power supply to constant current mode with the current set to 350
mA.
vii. Continue growth for 60 min to produce 6-μm-long fibers.
viii. Terminate growth by turning off the high voltage.
ix. Turn off the gas flow, open the pressure control valve to pump
down the chamber, turn off the heater, and wait until the wafer is
cooled down to at least below 300°C before removing.
16. Spotting of Nanofiber Arrays with
Plasmid DNA
■ For DNA delivery applications, VACNF arrays must be surface
modified with DNA prior to cellular interfacing. VACNF arrays may
be spotted with DNA or DNA may be covalently linked to the
nanofibers.
■ The former procedure relies on physiadsorption of DNA on the
nanofiber surface during spotting, and release of this DNA once the
nanofiber has been introduced via penetration into the intracellular
domain.
■ Nanofiber surface composition is an important factor that influences
both adsorption and release of material. We have found that bare
carbon nanofibers are poor DNA carriers. They are typically
hydrophobic and, thus, do not wet easily with aqueous DNA
solutions.
17. Furthermore, DNA is only weakly held on the surface of bare carbon
nanofibers, apparently often being shed prior to introduction of the
nanofiber within a cell. By contrast, nanofibers that are synthesized
under conditions that redeposit silicon nitride on the sidewalls of the
growing structure are found to promote strong adsorption of DNA
during spotting but release at least some of this material once the
structure is introduced into a cell.
The specific steps for spotting DNA on VACNF arrays are as follows:
1. To promote wetting of prepared nanofiber arrays, expose the array
samples to a 30-s radio-frequency (RF) oxygen plasma etch process
(RF power = 115 W, pressure = 350 torr, oxygen flow rate = 50
sccm).
18. 2. In a chemical fume hood, cleave large samples into 5-mm2 chips using a
diamond scribe to make straight cleavage lines across the surface being
broken. Snap at the scored line by placing the line directly over a rigid,
straight edge and tapping the side to be broken at an edge, not on the
fibered surface.
3. Following all cleavage procedures, rinse the samples in a spray of distilled
water to eliminate debris.
4. Spot 5-mm2 fiber arrays samples with 1 to 2 μL of plasmid DNA at
concentrations of 10–500 ng/μL. Ideally, plasmid DNA should be
suspended in water, as opposed to conventional buffering solutions, in
order to avoid the formation of salt crystals during the subsequent drying
step.
5. Air-dry each spotted sample in a sterile culture hood. Typically, this takes
approx 10 min under normal laboratory humidity.
19. Covalent Modification of Fibers With
Plasmid DNA
■ To provide more control over the fate of introduced genes than that
provided by the physisorption/desorption mechanism of spotting,
delivered material may be physically tethered to the nanofiber scaffold.
■ Covalent attachment of plasmid DNA is achieved using a condensation
reaction between primary amines of DNA bases and carboxylic acid
sites on the nanofiber surface.
■ Using this method, the DNA may attach at multiple locations, and the
sites of attachment within the DNA cannot be specified.
■ As such, it is likely that much of the plasmid DNA will be rendered
transcriptionally inactive, because attachment at sites within the active
coding region of the plasmid will interfere with polymerase access and
function.
■ Nonetheless, this technique has provided transcriptionally active,
bound plasmid using a 5081-bp plasmid with a 30% active coding
region
20. Fluorescent
image of Green
Fluorescent
protein (GFP)
Fluorescent micrograph of
green fluorescent protein (GFP)
expression in CHO cells 1 d
after integration with fiber
array covalently derivatized
with pgreenlantern-1, a GFP
reporter plasmid
21. For highest yield, it is recommended that plasmid DNA be suspended
only in water. Both Tris and EDTA of TE DNA buffers contain reactive
groups that will interfere with the EDC condensation reaction of DNA
onto the carboxylic acid sites of fibers.
The specific steps for covalent attachment of DNA to VACNF arrays
are as follows:
1. Provide or increase the number of carboxylic acid sites on the fiber
surfaces by exposing array samples to a 5-min RF oxygen reactive ion
etch (RIE). A typical oxygen RIE recipe for a Trion etcher is a pressure
of 350 mt, an RIE power of 115 W, and an oxygen flow of 50 sccm.
This step may be conducted on either discrete samples or large (wafer-
scale) nanofiber arrays.
2. In a chemical fume hood, cleave large samples into 5-mm2 chips using
a diamond scribe to make straight cleavage lines across the surface
being broken. Snap at the scored line by placing the line directly over a
rigid, straight edge and tapping the side to be broken at an edge, not on
the fibered surface.
22. 3. Following all cleavage procedures, rinse the samples in a spray of
distilled water to eliminate debris.
4. Place individual 5-mm2 array samples into 1.5-mL Eppendorf tubes,
taking care to handle the samples by the edges. The curve of the
Eppendorf tube will protect the fibered surface from contact with the
tube walls.
5. Dispense 500 μL of 0.1 M MES, pH 4.5 buffer containing 10 mg of
EDC to cover each fibered sample in its reaction tube. Ensure that the
fibered surface is wetted and remains submerged and does not harbor
trapped air bubbles.
6. If a control sample is desired in order to evaluate the effects of
covalently attached vs nonspecifically adsorbed DNA on fibers,
dispense 500 μL of 0.1 M MES, pH 4.5 buffer containing noO EDC to
cover the control samples in their reaction tubes. Ensure that the
fibered surfaces are wetted and remain submerged and do not harbor
trapped air bubbles.
23. 7. Add 1 μg of plasmid DNA in water to each sample, triturating the
dispensed fluid to disperse into the solution.
8. Agitate these reaction tubes on an orbital shaker for at least 2 h at
room temperature, ideally overnight, to allow the reaction to run to
completion.
9. Aspirate each reaction mixture with a Pasteur pipet, taking care not to
touch the fibered surface.
10. Rinse each reaction tube in two, 1-mL aliquots of PBS and then soak
in 1 mL of PBS for 1 h at 37°C.
11. Rinse each reaction tube in two, 1-mL aliquots of deionized water.
12. Dry each sample prior to use.
24. Preparation of Microcentrifuge Spin
Tubes
■ For some cell types, fiber penetration into a cell may be achieved by
centrifuging the cells down onto the vertical array of nanofibers. A
modified Eppendorf tube may be implemented for rapid cell-fiber
interfacing with a benchtop microcentrifuge.
■ Because microcentrifuges typically employ rotors that hold tubes at a
45° angle, the modified Eppendorf tube is designed to feature a 45°
slanted surface such that a small (approx 5 × 5 mm) VACNF array
chip may be positioned on the slant normal to centrifugal force.
25. The specific steps for constructing a modified Eppendorf tube are as
follows:
1. Wearing appropriate chemical protection gloves and glasses, prepare
approx 10 mL of PDMS by mixing 10 g of component A (Sylgard 184;
Dow Corning) with 1 g of Sylgard 184 curing agent. Avoid generating
excessive bubbles during mixing by using a gentle folding motion with a
flat, stainless steel weigh spatula.
2. Place the mixed PDMS solution into a 10-mL syringe to allow
convenient dispensing of aliquots of the PDMS solution.
3. Dispense 0.5 mL of the PDMS solution into each 1.5-mL Eppendorf
tube to be modified. Cap each tube after filling.
4. Place the filled Eppendorf tubes in a microcentrifuge, carefully rotating
each tube so that each tube’s lid hinge is positioned pointing away from
the centrifuge rotor’s center. This positioning will be used each time the
spin tube is implemented in order to orient the tube properly.
26. 5. Spin the centrifuge for 12 h at 2000g and ambient temperature. If the
centrifuge features a heater, cure time may be reduced to 30 min at
65°C. During this time, the PDMS in each tube will slant and cure,
forming a semirigid, planar surface normal to the centrifuge’s radial
vector.
6. Open each tube, place the tubes in a beaker, cap the beaker with
aluminum foil and autoclave indicator tape, and sterilize the lot in an
autoclave.
7. Following sterilization, dry any excess moisture by placing in a drying
oven (temperature not to exceed 95°C).
27. Interfacing of Cells Onto Carbon
Nanofiber Arrays
Fiber penetration and material delivery into a cell appears to be a
multistep procedure. For small dye molecules, simply centrifuging the
cell onto fibers for brief intervals (minutes) at high pelleting forces
(approx 1000g) can provide cell loading.
For DNA delivery, centrifugation and a subsequent press step is much
more effective than centrifugation alone, perhaps providing improved
penetration of nanofibers across the nuclear membrane barrier (Fig).
Although some success at cell-fiber interfacing has been achieved
simply by allowing cells to settle out of suspension onto fibers and
then performing a press, interfacing effectiveness and material delivery
to the nucleus is improved by first centrifuging cells onto the fibers.
Centrifuge parameters will likely vary for different cell types, and
these should be adjusted and optimized appropriately.
28. For CHO cells, pelleting forces of 600g for 30 s to 1 min are effective
if followed by a subsequent press step.
Following centrifugation of cells onto fibers, a subsequent press step
dramatically improves fiber penetration into the intracellular domain.
This press step should be performed on a relatively flat surface (<1
μm surface roughness) that is compatible with the cells being studied.
Ideally, to increase the effectiveness of the press step for the entire
chip surface, the surface should be somewhat flexible and compliant.
An autoclavable dish that has been partially filled with PDMS, as
described next, works well as a stamping pad.
29. a. Preparation of Sterile, Compliant
Pressing Surface
1. Wearing appropriate chemical protection gloves and glasses, prepare
approx 2 mL of PDMS for each stamping pad to be used by mixing 2 g
of component A (Sylgard 184; Dow Corning) with 0.2 g of Sylgard 184
curing agent. Avoid generating excessive bubbles during mixing by
using a gentle folding motion with a flat, stainless steel weigh spatula.
If excessive bubbles are formed during mixing, place the mixture in a
centrifuge tube and spin at more than 100g for 5 min. This will
effectively remove air bubbles from the mix.
2. Pour the PDMS mixture into an autoclavable dish (more than 35 mm in
diameter) such that at least 1 mm of PDMS solution covers the entire
bottom of the dish.
3. Cure the PDMS by placing the dish on a flat surface at 65°C for at least
30 min.
4. Autoclave the stamping dish to sterilize.
30. Mouse myeloma cell (SP2/O-
AG12) cultured for 3 d after
centrifuging and pressing onto
a 5-μm spaced nanofiber
forest. This cell grows in
suspension culture and does
not attach and stretch out onto
the fibers or fiber substrate
SEM images:
Cultured Mouse
myeloma cell
31. b. Optimization of Centrifugation
Parameters
■ Prior to interfacing cells to nanofiber arrays, cells must be suspended in
serum- and dye-free buffer solutions.
■ Medium constituents, and particularly serum, can have mitogenic
effects on cells if administered directly to the intracellular domain.
■ Thus, for all cell-interfacing procedures, cells should be washed of their
medium and resuspended in PBS, or other buffers appropriate to the cell
being studied.
■ Centrifugation parameters can be optimized by using a membrane
impermeant DNA intercalating stain to monitor membrane rupture (an
indicator for fiber penetration) and membrane resealing (required for
cell recovery).
■ Ideal centrifugation parameters will result in a high probability of
membrane rupture, but also a high probability of membrane resealing,
such that manipulated cells remain viable after the interfacing
procedure.
32. These tests can be performed using centrifugation trials with a
membrane-impermeant stain in solution during centrifugation (to
evaluate membrane rupture), and with this stain added to solution
approx 5 min after centrifugation (to evaluate membrane resealing or
general cell recovery). Propidium iodide (PI) and ethidium homodimer
are both effective impermeant dyes (Figure).
Both, however, are suspected mutagens and should be handled with
caution.
The specific steps for optimizing centrifugation parameters are as
follows:
1. Using appropriate laboratory safety procedures, prepare a 1 mM stock
solution ofeither PI or ethidium homodimer.
2. Prepare cells for interfacing by suspending adherent cell types
(scraping or trypsinization as appropriate) and washing all types free
of medium, dyes, and serum using pelleting and resuspension.
Resuspend in a cell-specific buffer, such as PBS, at a dilution that will
provide the desired monolayer coverage for a 1-cm2 surface area using
0.5 mL of cells. Approximately 30 mL of cells will allow 10 different
centrifugation trials.
33. (A) When cells are centrifuged
onto fibers in the presence of
impermeant PI, the dye passes
the plasma membrane and
stains DNA/RNA within the cell.
(B) If PI is added to the system 5
min after the centrifugation
procedure, it is excluded from
the intracellular domain,
apparently owing to resealing of
the plasma membrane following
fiber penetration.
PI uptake in cells
centrifuged onto
nanofiber arrays
34. 3. In the following steps, work with only one set of samples at a time for
each spin setting to be evaluated. Each set will consist of three samples
with dye in the solution during the spin, and three samples with dye
added to the solution after the spin. Label these P1, P2, and P3 for
“penetration,” and R1, R2, and R3 for “resealing.”
4. Place the fibered samples into the PDMS slant of modified Eppendorf
tubes such that the unfibered back side of the substrate is flat on the
PDMS surface in the center of the tube.
5. Triturate the cell suspension to resuspend the cells before each trial, and
place 0.5 mL of the cell suspension in each sample tube.
6. Add 5 μL of the 1 mM solution of dye to three of the samples labeled P1,
P2, and P3. Triturate to mix.
7. Close all the samples and load into a microcentrifuge. Ensure that the
samples are loaded opposite to one another in order to balance the
microcentrifuge. Also ensure that the lid hinge of each tube is positioned
pointing outward from the center of the centrifuge (as they were
positioned during fabrication) such that the slant of the PDMS is
oriented appropriately. The slant of the PDMS should be oriented
straight up and down when loaded correctly.
35. 8. Spin for the desired force and time. Trial 1 should be set at a time and
force typical for gentle pelleting of the cell line studied. Subsequent
trials will increase either or both of these parameters.
9. Following the spin, wait 5 min.
10. Carefully aspirate the solution from the P1, P2, and P3 samples and
carefully add 0.5 mL of neat PBS.
11. Add 5 μL of the 1 mM solution of dye to the three samples labeled R1,
R2, and R3.
12. Incubate for 5 min plus the centrifugation time, in order to incubate the
R samples for the same amount of time the P samples were incubated
with dye.
13. Carefully aspirate the solution from the R1, R2, and R3 samples and
carefully add 0.5 mL of neat PBS.
14. Remove the samples from all six modified Eppendorf tubes, and
observe the surfaces of each sample with an epifluorescent microscope
equipped with a TRITC filter set (535/610).
36. 15. Repeat steps 4–14 until an optimized centrifugation protocol is
achieved in which P samples result in high numbers of dyed cells
(indicating potential penetration by fibers into cells), but R samples
maintain very low numbers of dyed cells (indicating resealing of the
membrane following puncture).
Note that depending on the cell line, culture conditions, and harvesting
techniques, membrane penetration and resealing may not be mutually
obtained. Ultimately, to achieve puncture merely using centrifugation,
pelleting conditions may be too extreme to allow cell resealing and
recovery. In this case, a more gentle spin protocol should be combined with
a subsequent press step to provide potential DNA delivery.
37. c. Increasing of Fiber/Cell Integration
by Pressing
■ If the press step is employed, it must be implemented as quickly as
possible following centrifugation in order to minimize additional
trauma to the cells that may result owing to membrane attachment to
the fiber surface and subsequent shear of these attached domains
during the press step.
38. ■ The specific press steps are as follows:
1. Immediately following centrifugation, carefully remove the chip from
the spin tube with fine-nosed tweezers. Grasp the chip by its edges with
the tweezers. Do not clasp the fibered surface.
2. Gently place the chip face down on the PDMS stamping pad, and
gently press the back surface of the chip using a force approximately
equivalent to writing with a pencil. Minimize lateral movement of the
chip on the PDMS surface so as not to shear fibers and cells from the
substrate.
3. Immediately remove the chip from the PDMS surface and place face
up in a culture dish.
4. Place sufficient buffer solution in the culture dish to submerge the chip.
5. Allow the cells to recover in buffer solution for at least 15 min.
6. Aspirate the buffer solution from the culture dish and replace with
growth medium.
7. Incubate under appropriate growth conditions for the cell line being
studied.
39. Notes
■ The procedures discussed describe the steps for delivery of material, in this
case plasmid DNA, into cells. There are many other potential applications of
functionally integrated nanostructures into viable cells that can be realized if
more extensive postgrowth processing of the VACNFs is performed. In
essence, the VACNF is a high-aspect-ratio, mechanically and chemically
robust conductor of electrons that can be deterministically produced on any
substrate compatible with the PECVD growth process.
■ Use of large-scale growth reactors created the opportunity to synthesize high-
quality VACNFs in precisely defined locations on substrates compatible with
microelectronic device manufacturing equipment (e.g., 100-mm-diameter
round Si and quartz wafers).
■ Consequently, it was discovered that VACNFs are compatible with many of the
standard microfabrication techniques used in the production of integrated
circuits and microelectromechanical systems. Although a comprehensive
description of microfabrication techniques for VACNF substrates
Timothy E.,McKnght, et al. NanoBiotechnology Protocols, Methods in Molecular Biology; Vol 303
40. ■ Some of the more useful processing steps:
a. Most fundamental operation is the deposition of thin films on VACNF arrays.
Examined the effect of SiO2 and amorphous Si deposition onto VACNF using RF
PECVD. These layers could be uniformly deposited onto the fibers, resulting in a
conformal coating. Physical vapor deposition techniques including sputtering and
electron beam evaporation have also been used successfully for this purpose. This
provided us with a mechanism to modify the surface of the VACNF. Of particular
interest was the coating of dielectric layers onto the VACNF that could be
selectively removed from regions of the fiber with subsequent microfabrication
processes. This provides the ability to control the amount of the fiber body capable
of participating in electron transport independent of the aspect ratio or geometry
of the fiber.
b. Once material has been deposited onto a substrate, typically some sort of
patterning is performed. Photolithography has long been established as the
standard workhorse technique used in the microelectronics industry for this
purpose. This process involves the patterning of ultraviolet–sensitive polymer
layers (photoresists). Photoresists are typically spin cast onto substrates at speeds
ranging from 1000 to 6000 rpm. Simple experiments involving the deposition of
photoresist layers with a thickness between 200 nm and 2 μm demonstrated that
even high-aspect-ratio VACNFs survive this processing. Not only can a photoresist
be applied to substrates with VACNF on them, but it can be exposed and developed
using well-established techniques.
41. Moreover, a photoresist can be stripped from substrates containing fibers using a
combination of organic solvents and ultrasonic agitation with no damage to the
structural integrity of the VACNF.
c. Once a pattern has been exposed in a layer of photoresist and developed, it is
transferred into the substrate by either the addition or removal of material, referred
to as additive or subtractive pattern transfer, respectively. In the case of subtractive
pattern transfer, some form of etching is used to remove the desired layer or layers
from the patterned area. These processes include various forms of plasma-based
etching along with wet chemical etching. Material deposited onto a VACNF can be
removed using several combinations of these techniques without significantly
damaging the fiber. The fact that the VACNF is composed of graphitic carbon
provides it with a robust body that can withstand bombardment of ions during
plasma-based processes and any sort of chemical reaction with the exception of
those designed to attack carbonaceous materials.
d. The processing techniques just described have resulted in the fabrication of several
microscale structures that exploit the unique nanoscale properties of the VACNF. A
process for passivating the body of the fibers with an insulating thin film while
leaving the tips electrically and electrochemically active was developed for the
fabrication of electrochemical probes with high spatial resolution (Fig.). This
technology was then combined with a process for creating individually electrically
addressable VACNFs on an insulating substrate to produce arrays of high-aspect-
ratio electrochemical probes
42. (A) VACNF grown on Si passivated
conformally with thin film of SiO2;
(B) Array of passivated VACNF
following spin coating of Photoresist;
(C) Array after a brief RIE, liberating
tip while leaving body passivated;
(D) Scanning transmission electron
micrograph of passivated VACNF
following tip release described in (C).
The VACNF tip extends beyond the
oxide sheath, producing a
nanometer-scale electrochemically
active surface.
VACNFs nanoscale
images: Fabrication
of electrochemical
probes with high
spatial resolution
43. e. Chemical mechanical polishing is frequently used in microelectronic circuit
manufacturing to planarize substrate morphology. This technique can also be
applied to films deposited onto VACNF. Conformal layers of SiO2 have been
successfully planarized without damaging the VACNF. Continuation of this
process has been shown to remove sections of the VACNF at the same rate as
the SiO2, leaving the exposed fiber core coplanar with the surrounding oxide
topography. This strategy has enabled the synthesis of coplanar electrode arrays
for electrochemical applications in which many fibers perform transduction in a
parallel fashion.
f. The VACNF can also be used as a sacrificial template for the creation of
vertically oriented nanofluidic devices. In this process, arrays of VACNF are
grown on either Si or Si3N4 membranes. The fibers are coated with a thin
conformal film using PECVD or low pressure chemical vapor deposition
(LPCVD). The tips of the fibers are liberated using the process described in
Note 1.d. for the individual electrochemical probes. Following removal of the
remaining photoresist, the substrates are subjected to a brief etch in nitric acid
to remove the Ni catalyst particle at the tip. An O2 RIE is used to remove the
body of the fiber from the thin film tube encasing it. Once the body of the fiber
has been entirely removed, the bottom of the tube can be opened, creating a
nanometer-sized pipe structure, or nanopipe (Fig). These types of structures
have been used in fluid transport experiments involving DNA and fluorescent
intercalating dyes.
45. The combination of the growth, postgrowth processing, and interfacing techniques
described in this chapter provides a set of unique tools for direct interaction with
cellular processes at the molecular scale. There is a great deal of promise in the
application of such high-volume, yet precisely engineered devices to problems of
biological interest. Perhaps as these tools evolve further, they will eventually be as
significant within biological fields as they have been for semiconductor electronics.
46. Conclusion
■ Arrays of Vertically Aligned Carbon Nanofibers (VACNFs)
provide structures that are well suited for the direct integration
and manipulation of molecular-scale phenomena within intact,
live cells.
■ VACNFs are fabricated via a combination of microfabrication
techniques and catalytic plasma-enhanced chemical vapor
deposition.
■ Synthesis of VACNFs and detail the methods for introducing
these arrays into the intracellular domain of mammalian cells for
the purpose of delivering large macromolecules, specifically
plasmid DNA, on a massively parallel basis.
Timothy E.,McKnght, et al. NanoBiotechnology Protocols, Methods in Molecular Biology; Vol 303