nano-101-lecture 12 (1)
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nano-biotechnology notesTRANSCRIPT
LECTURE 12
NANOBIOTECHNOLOGY Biotechnology Meets Nanotechnology
A) Why? B) Biomolecules building blocks C) Nanomaterials-Biomolecules Hybrids D) Applications (Nanomedicine, Nanosensors, Nanomachines, Nanofabrication)
Nano 101 Introduction to NANOTECHNOLOGY
• Content: Introduction • What is nanotechnology? • History of Nanotechnology • Introduction to Miniaturization • Understanding Size • Nanoscale Physics • Scaling Laws • Photonic properties of Nanomaterials • Nanoscale Heat Transfer • Nanoscale Motion • Nanoscale Materials • Nanoparticles • Nanotubes and Fullerenes • Nanowires • Tools for Nanoengineering • Top-Down Nanofabrication Approaches • Bottom-up Nanofabrication Approaches • Nanoscale Characterization/Imaging (Scanning-probe microscopy) • Nanobiotechnology • Biological building blocks (DNA, peptides, etc) • Nanomaterial-Biomolecules Hybrids • Bio-inspired Assembly and Motion • Nanotechnology Applications • Nanomedicine • Energy (Fuel cells, solar cells) • Nanosensors • Commercialization efforts • Future Prospects
Exam 3: Wed the 26th (Tools in Nanoeng+Nanobiotech.)
Nanobiotechnology Biotechnology Meets Nanotechnology
Nanobiotechnology is the branch of nanotechnology with biological and biochemical applications.
Nanobiotechnology often studies existing elements of nature in order to fabricate new nanoscale devices.
Nanobiotechnology also involves using the tools, components and process of nanotechnology to biosystems.
Bionanotechnology: Creation of Nanobiomaterials
Bionanotechnology is a new frontier of research that combines two seemingly incompatible objects: the building blocks of life and synthetic nanostructures, both of them at a tiny, molecular-sized level. Its focus is on the development of powerful techniques and methods that merge the strengths of nanotechnology, working typically in the range of 1 to 100 nanometers, and biology, to generate a new type of 'bionanomaterial' which has some uniquely designed properties.
When combined with molecular biology, the possible applications of this applied nanoscience can become widespread and significant for example, nanomachines, biosensors, nanomedicine conjugated nanoparticles, and semi-synthetic nanopores it, once implanted in the human organism, could perform a number of medical and other functional duties, from delivering drugs to detecting malignant cells.
JW/Dept NE
THE SIMILAR DIMENSIONS OF NAOMATERIALS AND BIOMOLECULES ALLOW THEIR INTEGRATION
for the creation of bionanoscale devices
NANOBIOTECHNOLOGY
Combining the building blocks of life and synthetic nanostructures.
Nanobiotechnology Nanobiotechnology is also the nanotechnology that is present in living systems. It involves the molecular machinery of an organism. The cells machinery contains 4 nanoscale components: ● Amino acids (for making proteins) ● Sugars (for energy) ● Nucleotides (for storing information) ● Fatty acids (for structure)
Building Blocks of Life.
These can be combined with various synthetic nanoscale materials to create nanobioscale devices.
Biomolecules: Amino acids for making proteins
• Amino Acids - Molecules containing both amino
and carboxyl functional groups and a side group R. The basic formula NH2CHRCOOH.
• Peptide - A short chain of amino acids
• Proteins - Macromolecules composed of one or more polypeptides, each comprising a chain of amino acids, linked by peptide (C-N) bonds.
Amino Acids (AA)
An organic compound (of ca. 0.6 nm size) containing an amino group (NH2), a carboxylic acid group (COOH) and a side group R that
characterize the specific amino acid. All groups are attached to a single carbon atom.
IMPORTANT BUILDING BLOCKS OF LIFE
There are 20 different types of amino acids.
Amino acid Letter Codes
• Amino acid sequences can be written using either the 3-letter code or a 1-letter code. The exact formatting of sequences varies with the application; by convention single letter codes are always capitalized.
Here are the codes for each amino acid:
• Amino Acid 3 Letter Code and 1 Letter Code Alanine, Ala A ; Leucine, Leu L ; Arginine, Arg R; Lysine Lys K; Asparagine Asn N; Methionine Met M Aspartate Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Histidine His H Serine Ser S Isoleucine Ile I Threonine Thr T Glutamine Gln Q Tryptophan Trp W Glutamate Glu E Tyrosine Tyr Y Glycine Gly G Valine Val V
Biologists give amino acids a code letter, as for DNA. This is much easier than writing out the whole name each time.
Peptides Amino acids can be tied together by peptide bonds to form
peptide chains. The link between one amino acid residue and the next is called an amide bond or a peptide bond. To form a peptide bond, the OH of a carboxylic group of one AA combines with the H of the amino group of a second AA to form a C-N peptide bond and release water.
Species in which two amino acids are linked, called a dipeptide.
PROTEINS (and amino acids)
Proteins are polypeptide molecules (or consist of multiple polypeptide subunits). The distinction is that peptides are short and polypeptides/proteins are long.
The building blocks of a protein are amino acids. To build a protein we need to build a long chain of amino acids. Typical sizes of proteins are 2- 50 nm (e.g., 9 nm for albumin or 2 nm for insulin), with MW from 6000 to over a million.
Proteins fold nicely in 3D, depending on the order of the AA in their chains.
Proteins play crucial roles in almost every biological process. Each protein within the body has a specific function.
Every function in the living cell depends on proteins, from catalysis of all biochemical reactions (by enzyme) to recognition and
Immune protection (by antibody) and cellular transport (e.g., walking protein, kinesin). Enzymes are known to increase the rate of a biological reaction.
PROTEIN STRUCTURE The polypeptide chain is the primary structure of proteins. Proteins also have secondary and tertiary structures, where the they are held together by hydrogen or disulfide (S-S) bonds, respectively. The tertiary structure controls the shape (‘conformation’) of the protein.
The tertiary structure of a protein molecule, or of a subunit of a protein molecule, is the
arrangement of all its atoms in space, brought about via disulfide bonding
.
The primary structure refers to the sequence of the different amino acids of the protein.
Quartenary structure (conformation) involves the association of two or more polypeptide chains into a multi-subunit structure.
Secondary structure is "local" ordered structure brought about via hydrogen bonding
mainly within the peptide backbone.
Proteins fold nicely in 3D, depending on the order of the AA in their chains.
PROTEIN STRUCTURE (Cont.)
Protein synthesis involves two big steps called transcription and translation. Transcription involves the synthesis of an RNA transcript using the cell's DNA as a template, leading to messenger RNA(mRNA). In eukaryotic cells transcription happens in the nucleus. Once the messenger RNA is made it then is transported to the cytoplasm where the information contained in the mRNA is translated into a sequence of amino acids making up a polypeptide.
Protein Synthesis: Transcription and Translation
DNA stores the information for making proteins, with 3 bases in the sequence code for
each amino acid.
DNA as a Nanoscale Building Material DNA (duplex) Structure
The most striking feature of the DNA molecule is its
double helix structure, proposed by Watson and
Crick. According to this model the DNA molecule
consists of two coiled strands run in antiparallel
direction, and held together by hydrogen bonding
between complementary bases. A complete turn
covers a distance of 34A and includes ten base pairs.
It has a diameter of about 2 nm, a short structural
repeat (helical pitch) of about 3.4 nm and a
persistence length (a measure of stiffness) of around
50 nm.
Use of DNA for
bottom-up self-assembly nanofabrication
Use of DNA for creating microscale objects with nm resolution
NUCLEIC ACIDS
Nucleotides
The building blocks of DNA are its 4 nucleotides
molecules. Each nucleotide has 3 portions: a heterocyclic N-containing base, a 5-
carbon sugar (deoxyribose) and a phosphate group.
Joined together, the nucleotides form the DNA.
One of the four bases G, A, T, C (guanine, adenine, thymine, cytosine) is attached to
each sugar, leading to 4 different nucleotides.
G and A are purines while C and T are pyrimidines.
DNA Structure: DNA is a biopolymer. The monomer units of DNA are
nucleotides, and the polymer is known as a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group.
There are four different types of
nucleobases found in DNA: A is for adenine , G is for guanine ,
C is for cytosine and T is for thymine
DNA Denaturation and Hybridization When DNA is denaturated, the double helix structure is broken down. Usually heat is needed to break the hydrogen bonds and disrupt the stacking interactions. Such heat denaturation of DNA is called melting.
When the two single-stranded (ss) DNA strands are complementary, they bind together to reform the double helix (ds DNA) in a process called hybridization. Hydrogen bonding between complementary C-G and A-T base pairs holds two strands of DNA together to form a right-handed double helix.
DNA as a Nanoscale Building Material DNA as a Construction Material
The complementary base pairing (hybridization) of DNA holds great promise for bottom-up self-assembly nanofabrication with nm resolution.
Properly designed synthetic DNA can thus serve as a programmable glue (via specific hybridization of complementary sequences). Such self-assembly route can lead to ultrasmall features. Other attachment chemistries can be used for the directed assembly of other molecules (e.g., proteins) and nanobjects (e.g., nanoparticles).
JW/NE Dept
The specificity of the DNA base pairing not only determines the chemical foundation for genetics, but also confers predictable conformational properties that make it an attractive material for the assembly of highly structured materials with specific features.
DNA BUILDING BLOCKS - FABRICATION
The DNA-based self-assembly route can lead to ultrasmall features.
Use of the binding specificity and structural predictability of nucleic acids. Taking
advantage of the unprecedented recognition and
assembling properties of DNA.
DNA Nanotechnology
DNA as a Nanoscale Building Material
DNA nanotechnology provides, simultaneously, parallel and geometrically complex nanofabrication by making use of the binding specificity and structural predictability of
nucleic acids. In particular, the complementary base pairing (hybridization) of DNA holds great promise for
bottom-up self-assembly nanofabrication with nm resolution.
Use of DNA for
bottom-up self-assembly nanofabrication based on the recognition and assembling properties
of DNA.
Programmable self-assembly
of 4 x 4 tiles.
DNA as a Nanoscale Building Material Construction of microscale objects with nanoscale
feature resolution
DNA: A Key Player in Future Bottom-up Nanotechnology
DNA-based electronics is a prime example of an entirely alternative approach. Efficient attachment of
DNA to metal surfaces or electrodes is essential for charge-transport measurements, scanning tunneling
microscopy, and for fabricating devices and sensors. DNA nanotechnology will take advantage of the
unprecedented recognition and assembling properties of DNA. Researchers are confident that DNA-
based nanoelectronic devices will enable to reduce the size of the current silicon-based devices by
approximately 1000 times.
A four-arm junction made from four strands of DNA illustrates the principle that self-assembly can be controlled through design of the
component strands.
AFM images of tetragonal 2D arrays assembled from DNA cross structures
DNA ‘Lithography’
Folding DNA to Create Nanoscale Shapes and Patterns
Towards DNA origami
DNA Origami
DNA Origami
The synthetic technique of DNA origami, involving a single scaffold strand being folded into arbitrary shapes through the attachment of smaller staple strands to various locations along its sequence, has provided a powerfully programmable framework for the spatial design of nanostructures.
Folding DNA to create nanoscale shapes and patterns.
What is DNA Origami?
DNA origami involves taking a long, single-stranded DNA backbone (usually about 7000 bases in length) and forcing it to adopt an arbitrary shape using hundreds of short, single-stranded DNA oligonucleotides (each usually 20 to 50 bases long). Folding DNA to create nanoscale shapes and patterns.
In a process, DNA origami structures are typically assembled through a process of heat denaturation followed by gradual
cooling, in connection to crossover at certain locations.
DNA ORIGAMI Very complex shapes can be made from DNA origami, which is ssDNA which can be folded by short ‘clips’ of complementary DNA. These crossovers at specific points to give the structure some solidity. The twists and turns of the scaffold are then fixed in place by hundreds of shorter 'staple' strands, which hold the structure in place and prevent the scaffold from unfolding.
P. Rothemund, Nature 2006
Low cost production of nanostructures. Software available for designing basic
structures.
DNA Origami Folding ssDNA folded by short ‘clips’ of complementary DNA.
These crossovers at specific points to give the structure some solidity.
Folding DNA to Create
Nanoscale Shapes and Patterns.
DNA-Guided Assembly of Nanoparticles
Accurate positioning of nanoscale objects
Use of the biomolecular recognition properties of DNA to guide the
assembly of nanoparticles.
Accurate
positioning of nanoscale objects. Control of the particle
assembled and the distance between these particles.
Eventually, assemble of multidimensional arrays containing nanoelectronic components with
the high-structural order.
JW/NE Dept
CARBOHYDRATES
Carbohydrates, or Sugars, have the general chemical formula (CH2O)n, where n can range between 3 to 8.
Carbohydrates consist of the elements carbon (C), hydrogen (H) and oxygen (O) with a ratio of hydrogen
twice that of carbon and oxygen. These molecules are the key energy source of our body,
providing 4 kilocalories (kcal) of energy per gram.
All sugars are very soluble in water because of their many hydroxyl groups.
Scientifically, sugar loosely refers to a number of carbohydrates, such as monosaccharides, disaccharides,
or oligosaccharides. Monosaccharides are also called "simple sugars," the most important being glucose.
Glucose: Glucose means sweet in Greek. Glucose has the molecular formula C6H12O6.
Glucose is by far the most common carbohydrate and classified as a monosaccharide. Cells use glucose as a source of energy and a metabolic
intermediate. When cells need energy, they break glucose to water and CO2, along
with ATP. The cells use the ATP for their energy needs.
Disaccharides are formed by two monosaccharides linked by a glycosidic bond, e.g., lactose:
The primary function of carbohydrates is for short-term energy storage; they act as fuels to enable use to carry out our physical activity.
Three common disaccharides: sucrose — common table sugar = glucose + fructose lactose — major sugar in milk = glucose + galactose maltose — product of starch digestion = glucose + glucose
Common monosaccharides: •glucose, "blood sugar", the immediate source of energy for cellular respiration. •galactose, a sugar in milk (and yogurt), and •fructose, a sugar found in honey.
CARBOHYDRATES (cont.)
Almost all sugars have the formula CnH2nOn (n is between 3 and 8).
Starch and cellulose are two common polysaccharides.
Cholesterol
Trigliceride
Oleate Acid
LIPIDS Lipids can be defined as natural substances that are insoluble in water but soluble in nonpolar organic solvents. Lipids are very diverse in both their respective structures and functions.
Phospholipids
A fatty acid is a carboxylic acid with a long aliphatic tail (chain). A phospholipid is formed when a pair of fatty acids are bonded
to glycerol which bonds to a phosphate group.
In water, phospholipids prefer a bilayer (sandwich) structure that holds together cellular membranes.
Major components of cell membranes.
Micelle
Liposome
Bilayer sheet
Cross-sectional views of the three structures that can be formed by
mechanically dispersing a suspension of phospholipids in aqueous solution
The red circles depict the hydrophilic heads of phospholipids, and the
squiggly lines (in the yellow region) the hydrophobic tails.
A micelle is an aggregate of lipid
molecules that arrange themselves in a spherical
form.
Nanomaterials-Biomolecules Hybrids
NANOBIOTECHNOLOGY (continue)
A) Why? B) Biomolecules C) Nanomaterials-Biomolecules Hybrids D) Examples and Applications
Integration of nanomaterial and biomolecules leads to novel
hybrid systems which couple the recognition or catalytic properties
of biomaterials with the attractive electronic and structural characteristics
of nanomaterials. Biomolecules and nanomaterials can be chemically
coupled by means of various methods.
BIOMOLECULES-NANOMATERIAL HYBRIDS
JW/NE Dept
Interfacing bottom-up chemical and biological assembly schemes with top-down
lithography to fabricate complex devices is presently a major goal in nanotechnology.
Interface between the top-down and bottom-up regimes.
THE SIMILAR DIMENSIONS OF NAOMATERIALS AND BIOMOLECULES ALLOW THEIR INTEGRATION
NANOBIOTECHNOLOGY
JW/Dept NE
BIOMOLECULES-NANOMATERIAL HYBRIDS
Several methods can be used for functionalizing nanomaterials with biomolecules. Depending on the specific nanomaterial, different functionalization schemes can be used for confining different biomolecules onto the surface. While molecular linkers (cross-linked to the captured biomolecule) are most commonly used, direct functionalization with the biomolecule can also be employed.
For example, alkyl thiols (SAM) bind strongly to gold particles or wires, histidine binds strongly to nickel particles, while isocyanides
bind strongly to platinum. Covalent coupling to nanomaterials can be accomplished
through a cross-linking agent (e.g., glutaraldehyde, amide) or in
connection to the end group of a thiolated monolayers (SAM).
Nanoparticle-Biomolecule Hybrids: based on different
interactions
Use of electrostatic
interactions, covalent
reactions, or bioaffinity
interactions.
RECEPTORS / LINKING AGENTS (Commonly conjugated with nanomaterials, e.g., for nanosensing or nanomedicine).
• Antibodies - Globular protein produced by an organism to bind specficity to foreign molecules, i.e., antigens. The remarkable selectivity of antibodies is based on the stereospecificity of the binding site for the antigen.
• Aptamers- Oligonucleic acids that bind a specific target molecule. Aptamers are usually created chemically by selecting them from a large random sequence pool (using the SELEX process). Unlike antibodies, aptamers offer long-term stability, target versatility, and convenient regeneration.
• Streptavidin- Tetrameric protein which binds very tightly to the small molecule, biotin. The binding constant for this interaction is very high (~10-15 mol/L) ranking among the strongest non-covalent interactions.
• Nucleic Acids- DNA hybridization.
Use bioaffinity interactions to form nanomaterial-biomolecules hybrids.
Nanobiosensors
Nanomaterials are of considerable interest for sensing applications
owing to their unique physical and chemical properties. Particularly
attractive for numerous bioanalytical applications are colloidal gold,
semiconductor quantum-dot nanoparticles or barcoded nanowires.
Such biosensors rely on coupling the nanomaterial transducer with a
biological recognition element (antibody, DNA probe, or enzyme). The
realization of effective nanobiosensors relies on the effective
immobilization of the biomolecule onto the nanomaterial transducer
to ensure high reactivity and accessibility, minimal non-specific
adsorption/background and high stability and speed.
Nanoparticle-based sensing protocols should have a major impact upon
clinical assays, environmental monitoring, security surveillance, or
food safety.
Nanoparticle-based DNA-Barcode Detection of Proteins
This powerful protocol relies on magnetic spheres functionalized with an antibody that binds specifically the target protein and a secondary antibody conjugated to gold nanoparticles that are encoded with DNA strands that are unique to the target protein.
C. A. Mirkin, Science 2003, 301, 1884.
AMPLIFIED MULTIPLEXED BIODETECTION OF PROTEINS
Change in optical properties resulting
from plasmon–plasmon interactions
between locally adjacent gold
nanoparticles. The characteristic red of
gold colloid has long been known to
change to a bluish-purple color upon
colloid aggregation.
In the case of polynucleotide
detection, mercaptoalkyloligonucleotide-
modified gold nanoparticle probes are
used.
DNA-INDUCED AGGREGATION of Au-NP and
RELATED COLOR CHANGES
BIOELECTRONIC DEVICES:
Nanowire-based magnetoswitchable tuning of bioelectrocatalytic processes.
Nanosensors and Actuators
Magnetic actuation triggering the biocatalytic reaction
and the detection of glucose
5 nm and 10 nm gold nanoparticles monofunctionalized with DNA were incorporated into triangular DNA tiles and assembled into a 2D array.
Directed assembly of nanoparticle arrays using DNA tiles
Nano Lett., 2006, 6, 1502
DNA base pairing has provided a “smart-glue” approach
Biomotor Imaging: Functionalizing quantum dots (QD) with active kinesin
biomotors and transport these dots along immobilized microtubules: Enable precise tracking of motors in vitro to understand motor stepping
and detachment under controlled conditions. Second, QD particles should enable individual kinesins to be followed
in cells, which is very difficult with current labeling procedures.
Third, QD can be used as models for biomotor-driven nanoparticle assembly in vitro.
Nanobio: Conjugating Quantum dots with Biomotors Semiconductor nanocrystals (quantum dots) have great
potential in biological imaging
Small. 2(2006)626
Nanowire Carriers for Drug Delivery
Perform simultaneously selective targeting, tailored therapy, and imaging functions.
Functionalized gold nanowires for in-vivo targeting of breast cancer tumors.
The wires are attached covalently to Herceptin through Nanothinks Acid.
PEG thiols are attached to the wire through their thiol moiety.