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.

Amino Acids and their Letter Codes (as for DNA)

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.

Nanosensors

Conjugating nanomaterials with bioreceptors

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

NANOSENSORS-Nanowire-based Detection of Viruses

Nanowire-antibody

hybrids for label-free

biosensing

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.

LECTURES 13

NANOMEDICINE