SOLUTIONS MANUAL for Bionanotechnology Concepts and Applications 1st Edition by Ljiljana Fruk, Anton

Chapter 1

1.1 1u = 1.660539040 × 10 − 27kg The mass of a silicon atom in kg is therefore: m = 28.05 × 1.660539040 × 10 − 27kg = 4.6578 × 10 − 26kg The volume occupied by a silicon atom: V=

m = 1.999 × 10 − 29m3 ρ

1.2 Assuming a transistor is a square sheet made of 10x10 atoms of silicon (radius of silicon atom is 110 pm), the surface area of this sheet is: Atransistor = a2 = (10 × 220 × 10 − 12m)2 = 4.84 × 10 − 18m2 N=

Achip Atransistor

= 1.24 × 1014

1.3 Au: 1 Cu: 1 Na: 1 C: 4 1.4. λ=

h = 7.27 × 10 − 9m me ve

1.5 The energy of the particle in the box can be calculated using the following equation E=

h2 2 n 8mL2

where h is the Planck constant (6.626 10-34 J s), L is the distance between the opposite walls of the box corresponding to the diameter of the particle, m is the mass of the particle and nis the energy level. ΔE =

(ni + 1 − ni)2h2 8mL2

= 1.5 × 10 − 18J

λ=

hc = 132 nm ΔE

1.6 Energy of the FCC (111) lattice: γ = 1.2 ε2 a Energy of the FCC (110) lattice: γ = 3.5 ε2 a

E (110) > E (111), the higher energy facet will be coated more quickly with the ligand X, because surface energy will be decreased upon ligand binding.

1.7 Eg = hν = 1.602 × 10 − 19J = 2.48eV 1.8 The surface area of the sphere: A = 4πr2 The volume of the sphere: V = 43πr3 2

The surface-to-volume ratio: AV = 4πr 3 = 3r 4/3πr

According to the above equation, the surface-to-volume ration is inversely proportional to the radius of the sphere, meaning that it will increase when the radius of the sphere decreases.

1.9 Ostwald ripening as well as coalescence/oriented crystal growth can be explained by taking into account the surface energy of the nanoparticle. High surface energy of smaller nanoparticles is responsible for their high instability and solubility. If we have a solution of smaller and larger nanoparticles, smaller nanoparticles will tend to dissolve into monomers, which can then be used for further growth of the larger nanoparticles..

Depending on the process, Ostwald ripening can either widen or narrow the crystal size distribution, and it slows down as the nanoparticle gets bigger.

1.10 Diffusion-controlled growth of nanoparticles can be explained using Fick’s law, which gives the relationship between the radius of a growing nuclei, time and concentration: J = 4πx2DdC dx , where J is the diffusion flux, D is a diffusion constant, and C is the concentration at distance x from the surface of nanocrystal.

Fick’s law indicates that in order to control growth, the concentration of the monomers should be kept low and the diffusion distance large. This can be achieved by changing the viscosity and concentration of the solution, or by introducing the surface barriers.

However, when diffusion is rapid, the limiting factor will be the kinetics of the surface reactions, addition and possible reduction of the monomers on the surface. In a kinetically or reaction-controlled growth of a nanoparticle, the rate will largely depend on the surface properties. Species attached to the surface such as various ions that either promote or block the reactions and the chemical reactions catalysed or quenched by the nanocrystal, will all affect crystal growth.

1.11 Quality of crystal seeds Experimental studies have shown that number of defects, such as those introduced by coalescence of differently oriented crystal facets, within crystal seeds can have a remarkable effect on the final shape of the nanoparticles. Such defects, which are a result of a fusion between different crystal domains, are often referred to as twin defects. In the case of metal and metal oxides, the shape of the seeds and the number of the twin defects will determine the final shape of the nanostructures. Those defects can sometimes be removed using oxidative etching using oxidative species.

Use of different surface ligands Compounds such as polyols can have different affinities to different crystal lattice facets and as they bind, they could arrest the growth alongside those facets enabling the controlled growth of the nanocrystal.

Chapter 2 2.1 Spherical gold nanoparticles: Turkevich method using sodium citrate as a reducing and capping agent and Brust-Shiffrin method.

Cubic gold nanoparticles: Turkevich method using citric acid as a reducing agent; seed-mediated growth using acidic reaction conditions and CTAC or PVP as capping agent.

2.2 Number of atoms in solution: N = cVNA = 6.022 × 1017 V

Number of Atoms in one NP: N = V NP

atom

The number of nanoparticles in the solution: N

N = Natoms in solution = 3 × 1012 nanoparticles atoms in one NP

2.3 (a) By increasing the concentration of starting materials (silver nitrate and formaldehyde) or by increasing the pH to shift the reaction equilibrium towards production of Ag. (b) Stable small nanoparticles can be obtained by decreasing the concentration of silver nitrate ions in the solution, slow addition of the reducing reagent or increasing the viscosity of the reaction solvent by addition of polymers such as polyethylene glycol (PEG). (c) Diffusion-controlled growth can be achieved by addition of viscous substances such as polymers (PEG, PVP).

2.4 The heating to 140°C is required for the hydrolysis of Ti(i-Pr)4 precursor.

The prolonged heating at 100°C improves the Ostward ripening process that leads to formation of rods.

2.5 In order to obtain smaller nanoparticles, the concentration of the precursor as well as the reaction temperature need to be kept lower. Another way to become smaller nanoparticles is to use a less reactive starting material, such as tetrapropyl silicate Si(OC2H5)4 with larger organic unit that makes it less reactive than smaller tetramethyl silicate Si(OCH3)4.

But there are also other factors that impact the nanoparticle shape and size such as the temperature and pH, as well as the presence of organic compounds, which can be

used to control the hydrolysis. In the case of SiO2 nanoparticles, reaction rate and size of the particles are strongly dependent on solvents, precursors, amount of added water, and the presence of bases such as ammonia. Ammonia is critical for the condensation step and it has been shown to aid the formation of the well-defined geometric shapes.

2.6 Shells such as zinc-sulfide (ZnS) are useful to prevent the leakage of toxic core elements such as cadmium (Cd) and selenium (Se) and smooth any crystal defects on the nanoparticle surface. The addition of a shell increases the overall size of the quantum dot, and since the fluorescence depends on the size (smaller quantum dots show higher fluorescence), only certain fluorescence emissions (larger wavelengths) can be achieved with core–shell systems.

2.7 Each carbon within the diamond structure engages in four bonds with neighbouring carbon atoms and it is sp3 hybridised. Atom geometry is tetragonal in diamond, and carbons form a perfect tetrahedral unit within the cubic cell. All electrons are localised and diamonds are generally bad conductors of electricity.

Fullerene has a large symmetrical structure composed of 60 atoms arranged into 20 hexagons and 12 pentagons. Carbon atoms in fullerene are sp2 hybridised, as they are in the graphite. However, the π- electrons are not delocalised between the layers as they are in the graphite, but rather within the carbon hexagons. Carbon nanotubes are similarly structured.

Carbon atom within a diamond has 4 neighbours, whereas in the case of graphite 3.

2.8 The synthesis of SWCNT is significantly improved by the addition of various metal powders such as nickel, cobalt, iron, tungsten and others, which act as catalysts. Metal atoms form alloy clusters, which can anneal all initially produced carbon structures into hexagons, and in such way promote the lengthening and orientation of the tube. Singlewalled nanotubes of controllable length, diameter, and chirality can be obtained using various catalytic clusters.

2.9 The surface area of the sphere: A = 4πr2 The volume of the sphere: V = 43πr3 2

The surface-to-volume ratio: AV = 4πr 3 = 3r ≈ 1 × 1010 4/3πr

2.10 V (Au) =

1g m = = 51.76 × 10 − 3cm3 = 51.76 × 10 − 9m3 ρ 19.32gcm − 3 V cube = a3, a = 3 V = 3.73 × 10 − 3m S cube = 6a2 = 8.34 × 10 − 5m2 5000m2 S(MOF) = = 59952038 S(gold cube) 8.34 × 10 − 5m2

Surface of one cube side: A = a × a = 1.39 × 10 − 5m2 −5 2

Thickness of the gold sheet with surface area of 5000 m2: c = 1.39 × 10 2 m = 2.78 × 5000m 10 − 9m.

Chapter 3

3.1 A cell membrane is a double layer of phospholipids and proteins that surround the cell creating a natural boundary between intracellular space (cytoplasm) and the extracellular matrix. Composed of two fatty acid chains (saturated and unsaturated) and glycerol–phosphate head, phospholipids can also contain additional functional groups, such as choline or serine, which determine their charge and the way they interact with neighbouring molecules. Presence of structural elements of opposing charge gives phospholipids an amphiphilic character meaning that they can be both hydrophilic (water loving) and hydrophobic (water repelling). In cell membranes, the phospholipid bilayer is arranged in such way that the hydrophilic heads are exposed to the exterior and interior of the cell, and hydrophobic tails are facing the membrane’s inner space. Most membrane proteins and many lipids contain covalently bound sugars (glycoproteins and glycolipids), which increase hydrophilic character of lipids and proteins and contribute to stabilisation of membrane protein structure. The membrane proteins can be found on the exterior and interior of the cell membrane (transmembrane proteins) and act as molecular gateways for control of molecular trafficking.

3.2 The cytoskeleton is a complex and dynamic system of filaments and tubules throughout a cell. A cytoskeleton supports the cell shape (and cells come in different shapes and sizes), guides the organisation of organelles, acts as internal cellular highway for transport of various building blocks and signalling molecules, and it is also involved in cell adhesion and tissue organisation. The main components of this important cell structure are three long filament structures: microtubules, linear actin filaments and intermediate filaments.

3.3 The mitochondrion. ATP is produced in a sequence of finely tuned chemical reactions known as the citric acid cycle or the Krebs cycle.

3.4 Lysosomes and peroxisomes are both membrane bound organelles found in the cytoplasm of eukaryotic cells. Both organelles are digestive vesicles with diameter ranging from 0.1 to 1.2 μm (peroxisomes tend to be slightly smaller than lysosomes) that destroy and remove compounds deemed unsuitable for further cellular use. .

Peroxisomes produce cholesterol and phospholipids found in brain and heart tissue.

Lysosomes are involved in various cell processes, such as plasma membrane repair, apoptosis, cell signalling and energy metabolsm. Lysosome’s interior is very acidic (pH 4–5) and contains many enzymes that help recycling and disposal of cell garbage.

Peroxisomes are oxidative organelles and are named after the large amount of hydrogen peroxide (H2O2) that they generate and scavenge. Besides several oxidases involved in production of hydrogen peroxide, peroxisomes contain catalase involved in the decomposition of hydrogen peroxide to oxygen and water.

3.5 (a) GUCGUCCUAAUG Amino acid sequence: VVLM; hydrophobic (b) AACCACAAA Amino acid sequence: NHK; hydrophilic (c) GCCACAUGG Amino acid sequence: ATW; intermediate

3.6 Tryptophan (Trp) and methionine (Met) are encoded by a single triplet.

Both are hydrophobic.

3.7 Based on the assumption that all proteins have approximately the same density of 1.37g cm-3, a simplified formula can be deduced, which states that M (in Da) = 825 V (nm3 ). For spheric proteins, protein radius can be calculated from the volume.

M = 50000Da, ⇒V =

50000 = 60,6 nm3 825

4 V sphere = πr3 3 r=3

3V 4π

= 2,43 nm

3.8 Antibodies can have up to 10 sites for antigen binding, which can vary in strength, but still enable tight overall binding. These are found at the tips of the two arms, in a pocket formed between light and heavy chain, and have very different lengths and amino acid composition.

3.9 The immune system of a treated patient mistakes the antitoxin for harmful antigen and initiates an inflammatory response.

Antiserum sickness can be developed as a result of exposure to the antibodies derived from animals. To avoid it, it is often best to use an antiserum produced from the same species.

3.10 Due to its structure and bad accessibility of recognition sites for the immune cells, collagen has a low antigenicity.

3.11 An enzyme cofactors are organic or inorganic compounds, which are either directly responsible for substrate binding or involved in electron transfer necessary for reactions to occur. Organic cofactors, such as widely found heme and flavin, can be covalently or non-covalently bound to the enzyme.

3.12 Collagen, elastin, keratin and fibroin are structural proteins responsible for structural integrity of the organism and also act as structural components to build protective external layers.

Collagen is composed of three interwoven polypeptide that form triple helices of different lengths, some of which are up to 420 nm. Triple helices further assemble into collagen fibres, which provide tensile strength to ligaments, bones and dense connective tissue and as such, reinforce most organs. Characteristic collagen triple helices contain a high percentage of glycine, proline and hydroxyproline, and the elasticity of coiled elastin regions stems from glycine–valine–proline triplets.

Keratin is a hard structural component found in humans and across the animal kingdom. Keratin has a high content of cysteine, which can form strong covalent bonds through sulphur–sulphur interactions (formation of disulphide bridges), leading to exceptionally robust structures. α-Keratin, a helical, fibrillary form of keratin, is a structural element of skin, hair, nails, horns and claws, whereas sheet-forming βkeratin can be found in birds’ feathers and claws as well as in most reptiles.

Silk fibroin fibrils contain distinct crystalline regions composed of mixed helices and sheets, and amorphous, flexible chains. Similar to other structural proteins, silk fibroin also has a high glycine content. However, similarity stops here since the fibroin fibres are composed entirely of protein sheet layers and lack any helical regions.

3.13 B-DNA is a right-handed helix with pitch of 10.4 bases (3.32 nm) and diameter of approximately 2 nm. Water molecules play an important role in the stabilisation of the helix by enforcing base stacking, and there are at least 12–15 water molecules associated with each nucleotide in B-DNA. A-DNA is a collapsed form of B-helix which happens when the humidity conditions change, bringing the phosphate groups closer together and changing the geometry of the structure. Unlike A- and B-DNA, Z-DNA, has a pitch of 12 bases, which makes it slimmer than B-DNA and it also lacks major grooves. Due to its structure, Z-DNA is less stable than other DNA forms and it needs to be stabilised with lots of salt. It is also considered a transient structure, which can occasionally be induced by certain biological processes, but disappears quickly.

Chapter 4 4.1 Dynamic self-assembly: Nanoparticle growth Magnetosome formation within magnetotactic bacteria Formation of a bacterial colony Concentric rings of charged polymer nanoparticles in electric field

Static self-assembly: Formation SAMs of thiols on gold surface Formation of single-walled carbon nanotubes Viral shell formation Assembly of metal-organic frameworks (MOFs) Assembly of a ribosome structure Static self-assembly refers to a formation of ordered structures, which reach an equilibrium state and do not change further following the assembly.

Dynamic self-assembly results in a non-equilibrium state and requires a continuous input of energy to maintain a steady state once the structure is formed. In the absence of this energy, the system falls apart. Energy is supplied either as heat, through irradiation or by application of a magnetic or electric field.

4.2 How could you modify the surface of CdSe nanorods to induce a self-assembly into chains of nanorods?

Surface ligands should be added that are capable of interacting with each other through weak forces. For example, single stranded DNAs can be added to the surface, or lipids.

4.3 Due to the amphiphilic character of the phospholipid membrane, the Au nanoparticles can be modified either with charged groups such as alcohols or amines or with lipophilic groups such as alkyl chains or cholesterol in order to interact with the cell membrane.

4.4 (a) OH-containing molecules, silanes (b) OH-containing molecules, silanes

(c) sulphur-containing molecules such as thioalkanes (d) Nitrogen oe sulphur-containing molecules such as lipoic acid or amines

4.5 Usually, in order to bind antibodies to other molecules, lysine residues of an antibody are coupled with a carboxylic acid derivative of the molecule of interest via amide bond formation. To modify graphene with antibodies, carboxylic acid groups need to first be introduced to graphene i.e by reacting graphene with a diazonium salt.

4.6 The wetting properties will be improved due to the hydrophilic character of carboxylate groups produced by oxidation of methyl.

4.7 (a) To make the nanoparticles soluble in water, hydrophilic molecules such as polyethylene glycol are used to exchange the hydrophobic non-polar ligands that makes them stable in organic solvent. (b) Since the catalysis in a large-scale industrial reactor requires stable nanoparticle-enzyme constructs, enzyme needs to be attached to the nanoparticle via strong covalent bond such as amide bond. (c) The nanoparticles should be functionalised to prevent aggregation, ensure biocompatibility and selective binding to the cancer cells, which need to be imaged.

4.8 The linker should contain a nanoparticle binding group such as hydroxyl or silane and an antibody binding group such as maleimide, which reacts with free cysteine. A spacer between the two reactive groups such as polyethylene glycol can also be used to reduce the steric hindrance and supress the unspecific binding of the antibody to the nanoparticle surface.

4.9.

Thiolated DNA can be directly attached to the surface of Au nanoparticles by chemisorption, due to the strong gold-sulphur interactions.

Maleimide modified DNA can be covalently attached to the tetrazole-modified Au nanoparticles via light-induced nitril-imine mediated tetrazole-ene cycloaddition (NITEC) reaction.

Amino-DNA can be coupled to lipoic acid coated Au nanoparticles Diels Alder reaction can be used between dienophile Au nanoparticle and diene DNA ( or other way around) ensuring that thiols are used for Au nanoparticle attachment.

4.10

One streptavidin molecule has 4 biotin binding sites. 20 streptavidin molecules can bind maximum 80 biotinylated DNA strands.

Chapter 5

5.1: (a) E = hν =

hc 6.63 × 10 − 34Js × 3 × 108ms − 1 = = 2.21 × 10 − 16J − 10 λ 9 × 10 m

E = hν =

hc 6.63 × 10 − 34Js × 3 × 108ms − 1 = = 3.75 × 10 − 19J − 9 λ 530 × 10 m

(b)

5.2 1 eV = 1.602 × 10 − 19J 100 keV = 1.602 × 10 − 14J E = hν =

hc hc , ⇒ λ = = 124 nm λ E

The electrons can be transmitted through a 0.6 μm silicon wafer due to their high energy. In general, high-energy electrons interact less strongly with matter than lower energy electrons, allowing the thicker samples to be explored. For the electron beam with an energy of 10keV the penetration depth through the silicon is around 1 μm, meaning that the electron beam with an energy of 100 keV can easily penetrate the silicon wafer of 0.6 μm.

5.3 Analyse the nanoparticles using X-ray techniques such as XRD (X-ray diffraction) or XPS (X-ray photoelectron spectroscopy).

5.4 In XRD, X-rays with wavelengths typically ranging from 0.7 to 2 Å generated by cathode ray tube, are filtered and focused onto a crystalline sample, and then diffracted by its crystalline phases. The diffraction peaks are the result of the constructive interference of scattered X-rays In SAXS, X-ray photon beams are generated by particle accelerators known as synchrotrons. The beams are focused onto the sample using the collimation instruments, and the structural data is obtained from the angular distribution of scattered X-rays at small angles.

5.5 Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). (a) STM exploits the principle of electron tunnelling. The measured tunnelling current depends on distance between the surface and the tip. AFM is based on the measurement of the deflection of the tip as it moves over the sample. Unlike STM, which is based on a continuous readjustment of the tip to maintain a constant tunnelling current, AFM measures very small upward and downward deflections of the tip while maintaining the constant force of the contact. STM

requires both the conductive tip and the conductive samples, and it can also be used provide the information on topology and electronic properties. AFM can be used for non-conductive samples, soft samples and measurements in liquids, and provides conductive and thermal profile as well as the information on the adhesive force and the strength of molecular interactions. (b) Advantages of STM: atomic resolution, electronic properties of the surface, lifetime of the surface electrons. Can be used to assemble atoms on demand.

Disadvantages of STM: specific technique that requires a lot of skill and precision, requires very stable and clean surfaces, excellent vibration control and sharp tips, scanning speed is very low and scanning area small, it requires conductive surfaces and the tip.

Advantages of AFM: high resolution 3D surface profile, used to image hard surfaces in air or vacuum, it works in liquids, which is particularly well-suited to image biological samples. The preparation an AFM sample is very simple: there is no cutting, no coating, no drying, no fixation needed. Requires regular and reproducibly flat surface onto which the samples are deposited.

Disadvantages of AFM: low scanning speed and small scanning area. Direct contact to the sample is needed; the sample must be flat and fixed to avoid the sample displacement. The resolution depends strongly on the shape and the size of the tip.

5.6 The shape and size of the tip has a direct impact on the resolution of the image.

High resolution cantilevers are available with 1 nm radius tips.

5.7 The cantilever and the tips are usually made of silicon, or silicon nitride. These can be first oxidised to introduce OH-groups onto the surface and silane compounds, such as amino-silane can be used to introduce amine groups. Those can then be modified using amide coupling to carboxylic acid-DNA. Other interactions such as maleimidethiol and range of click chemistries could also be used providing that the modified DNA is available.

5.8 When a molecule absorbs energy, it can be excited to a higher energy state meaning the electrons jump to higher energy level. Those electrons can can transit to a lower energy state under emission of a light (luminescence, fluorescence, phosphorescence). This process is called spontaneous emission.

In case of stimulated emission, a photon is used to force an electron from the excited energy state to get back to the ground energy level while releasing an additional photon. As a result, two photons are emitted, an incident photon and the photon released when the electron moves to the ground energy level.

5.9 FTIR spectrometers provide better resolution and allow rapid scanning by allowing for the simultaneous collection of information from multiple frequencies.

5.10 All of the fluorescent proteins have a unique barrel-shaped structure, evolved to protect the inner core, which contains a chromophore composed of amino acids. This chromophore is produced in a series of chemical reactions catalysed by the protein itself (autocatalysis) and its optical properties are affected by the protein’s microenvironment.

5.11 The extinction coefficient could be increased by conjugation of the additional chromophores or some extended π-conjugated system to the GFP chromophore. The stability of the artificial system needs to be additionally ensured by mutation of the chromophore stabilising sites.

5.12 d = 4.7 nm 5.13 Characterisation of gold nanorods: TEM, DLS and Zeta potential in order to determine their morphology, size distribution and stability.

After functionalisation: the presence of the peptides could be investigated using XPS, DLS, Zeta potential.

Modification of the nanorods with an antibody: gel electrophoresis and protein staining Nanorod interactions with cells: AFM, QCM and if there is a facility available cryo-TEM.

5.14 UV–Vis spectroscopy will indicate the aggregation of the nanoparticles, but DLS could also be employed. To study the salt concentration that causes gold nanoparticles aggregation, nanoparticle solutions with different salt concentrations can be prepared and the UV-Vis spectra taken. A peak maximum shift to the longer wavelengths indicates the aggregation of nanoparticles.

5.15 Surface enhanced Raman Scattering (SERS)

5.16 Both are vibrational spectroscopy; IR based on direct absorption and Raman on inelastic scattering of photons. IR uses IR light, while in Raman range of wavelengths can be used. Vibration is Raman active if it accompanies the change in polarizability.

Ramans signals are very weak, but unlike IR Raman spectroscopy is not sensitive to the presence of water. Advanced IR and Raman spectroscopy provide nm-scale resolution when combined with AFM.

5.17 Dynamic light scattering (DLS)

5.18 FRET if the streptavidin is modified with appropriate acceptor dye, SPR studies

5.19. a) TEM, UV-Vis using known extinction coefficient tables b) Gel-electrophoresis, AFM, UV-Vis spectroscopy c) AFM, cryo TEM d) X-ray diffraction e) FRET providing we label the protein correctly f) hybridise the DNA with fluorescently labelled cDNA and use fluorescence microscopy, UV-Vis spectroscopy for DNA-protein conjugate g) XPS, EDX, TOF-SIMS

Chapter 6

6.1 V base pair = disc = πr2h = 1,06 × 10 − 27m3 4 V nucleus = πr3 = 1,41 × 10 − 17m3 3 N base pairs in a nucleus =

V(nucleus) = 1,33 × 1010 V(base pair)

⇒N bases ≈ 26 × 109

6.2 Hydrogen bonding between specific pairs of bases (A will bind to T and G to C),  - stacking and hydrophobic interactions between bases as well as electrostatic interaction between negatively charged phosphates and positively charged ions such as Mg2+.

6.4 200, each streptavidin has 4 binding sites for biotin molecule

6.5 (a) L stretched DNA = 3.5 × 109 × 350 × 10 − 12m = 1.23 m (b) L DNA of all cells = 1.23m × 50 × 1012 = 61.5 × 1012 m It would reach Pluto which is 5.91 × 1012m away

6.6 Preparation of antibody–DNA conjugates usually requires DNA strands to be modified with functional groups that can react with a native or chemically or geneticlly introduced reactive groups of an antibody. Such functional groups can either coordinate protein tags or bind covalently. Antibody Lys can be modified with COOHDNA via amide coupling. Cys can be introduced to the antibody and coupled with maleimide-DNA. Protein tags such as SNAP tag can be used and the small binding group attached to DNA to allow for coupling. Other strategies such as click chemistry can be used but require addition of appropriate groups both to antibody and DNA.

6.7 (a) 5‘-CAG GAT GAT ATT T (b) Two batches of nanoparticles need to be modified with short DNA strands with different sequences which are not complementary to each other, but have parts complementary to cystic fibrosis gene. When a target DNA (cystic fibrosis gene) is present in the solution, hybridisation of nanoparticle–DNA conjugates will lead to the assembly of gold nanoparticles, which results in the change of colour (red to violet) due to the interacting plasmons.

6.8 c Au NP =

0.3 4.2 × 108M − 1cm − 1 × 1cm

c DNA =

0.1 126000M − 1cm − 1 × 1cm

N DNA strands per Au NP =

= 7.14 × 10 − 10M

= 7.94 × 10 − 7M

c(DNA) = 1112 c(Au NP)

6.9 1. The origami platform needs to be designed to contain short single-stranded oligonucleotides (protruding arms) to allow for DNA-directed immobilisation.

2. Proteases need to be modified with DNA strands complementary to protruding arms using one of the strategies to design protein-DNA conjugates.

3. Immobilisation of proteases is achieved by mixing origami with protease-DNA conjugates.

Chapter 7

7.1 helix is helical structure composed of usually 7 amino acids, within which position 1 and 4 are usually hydrophobic amino acids while the rest is hydrophilic.

They are important components of fibrous proteins found in water-rich environments. sheets are planar structures composed of two strands composed of 5-8 amino acid residuse held together by a dense hydrogen bonds. In structural proteins these elements are responsible for toughness of the structure.

7.2 b) and c) are amphiphilic peptides. They usually contain a hydrophobic carbon chain which, in case of b and c is CH3-(CH2)6

7.3 hydrophobic-hydrophilic interaction, van der Waals interactions between the hydrophobic carbon chain and hydrogen bonds between sheet elements.

7.4 Cysteines contain thiol (-SH) group, which engages in formation of stabilising disulfide (-S-S-) bridges with other cysteine residues.

7.5 Carboxy PEG can be attached through amine group of lysine (Lys) residues, aspartic (Asp) and glutamic (Glu) acids contain carboxylic acid groups so would be the most suitable for direct amide coupling of amine PEG.

7.6. Phosphate groups are excellent coordinating agents for calcium ions (Ca2+) and eases calcium deposition and formation of more rigid, bone-like structures.

7.7. Nacre is made of layers of proteins and inorganic CaCO3 nanocrystals selfassembled into tablet-like structures, which stack into macrostructures. This results in a heterogenous composition and ensuring toughness not found in a brittle mineral aragonite.

7.8. Identification and isolation of proteins involved in biomineralization. Additional obstacle is posed by lack of understanding of the complex kinetics of the biomineralization processes.

7.9. Use of superhydrophobic surfaces inspired by lotus leaf; nanostructured coatings can be applied to prevent adsorption of bacteria and the growth of biofilms.

7.10 The isolated peptide was likely the one that binds to the plastic used to make the vials. Silica based structure for example, glass vial should be used and additionally treated to ensure that the surface is coated in such way to prevent the adhesion fo the phages.

7.11 Most likely cysteine-rich peptide formed disulfide bridges between different virus units creating larger assembles of viral particles, which then acted as templates for assembly of gold nanoparticles. Careful design of the peptide structures should be attempted to ensure that there is limited number of cysteines and they do not engage in formation of stable sulfur bridges.

7.12. Such packaging can be made of nanopatterned cellulose ensuring the appearance of non-fading structural colour. In addition, such structures could be modified with bacteria binding elements (a small peptide or a protein). As the bacteria bind, the pattern is disrupted inducing the changes in the light interaction and the change of colour.

7.13 butterfly wings, diatoms, insect shells, shrimp and moth eyes

Chapter 8

8.1 Biological recognition element that interacts with a target species, and a transducing element for signal detection. Nanobiosensors utilise nanomaterials incorporated into the design to enhance the sensor response.

8.2 Label-based: a), c), f) Label-free: b), d), e)

8.3. In order to design the electrochemical nanosensors it is important to ensure the communication between the biomolecule (enzyme) and the electrode. If the enzyme is linked through non-conductive linker poor electron transfer to electrode can be expected requiring use of mediators, on the other hand direct contact or use of conductive linkers will ease the transport of electrons and ensure the signal transduction.

8.4 If we only consider the length of CNT, and taking into account that one enzyme can be immobilised every 5 nm, then 100 enzymes would fit onto CNT. However, one needs to consider the geometry of the CNT and the protein: Considering that AB = 2.5 nm and AC=3.5 nm We get ½  = sin-1 (2.5/3.5) = 45.58, therefore  = 91.17 The number of proteins that can fit around the nanotube = 360/91.17 = 3.94 This number needs to be rounded to the highest reasonable number, which is 3 as you cannot force 4th protein onto the tube.

The number of proteins that can fit down the length of the nanotube = 500/5 = 100 Total number that can fit around CNT= 3x 100 = 300 proteins

8.5. Mediators act as efficient electron shuttles to ensure there is a read-out of the enzymatic reaction at the electrode. Suitable alternative to mediators are conductive nanomaterials such as metal nanorods or carbon nanotubes.

8.6. Refer to Chapter 5 for details on SPR principle.

Plasmonic nanoparticles can be used to enhance the plasmon density on the SPR chip and lead to increased sensitivity to the surface changes introduced for example by biomolecular binding or attachment of pathogens. In addition, due to their large surface area, nanoparticles also ensure that there is a larger area available for immobilisation of interacting species, or biomolecular recognition elements.

8.7. a) CdSe is used as FRET donor, EYFP as FRET acceptor in a FRET biosensor that can provide information of the distance between two FRET partners, and therefore signal when the nanobody has interacted with Rec 1 receptor. To ensure this, ReC 1 should be expressed to contain EYFP, and nanobodies should be attached to CdSe quantum dots. b) Rec1 can be labelled with EYFP using protein fusion design (Chapter 3), and nanobody can be attached to CdSe using either a suitable linker containing thiol groups for binding onto CdSe, and a functional group for further reaction with nanobody, or using cysteine (if available in nanobody) for direct immobilisation. c) Using EFRET =

1 and R0=9.78 d 1+ R 6

× 103(κ2n − 4QDJ λ )1 / 6 we get 5.61 nm as the

0

separation.

8.8. a) We need to detect the faulty gene of the CF patient. This can be done by designing two different batches of gold nanoparticles each modified with short DNA strand complementary to half of CF DNA, but not to each other. When there is no CF DNA present in the solution, gold nanoparticles will stay in solution. If CF DNA is present it will hybridise to nanoparticles bringing them together and creating networks, which will results in the change of colour.

D1 for gold NP1 and D2 for gold NP2 should contain a part of cCF-DNA ( 5’-ACG GAT GAT ATT T). b) UV-Vis spectroscopy would be suitable and the easier to use. c) Controls: use healthy patient DNA with both NPs, use one batch of NP containing random sequence, which is not complementary to CF DNA, also nanoparticles without DNA to see that there are no unwanted aggregation processes.

8.9. Proteases can be immobilised on the cantilever or QMB, and LF42 allowed to bind. As it binds, the change in mass will induce the change in frequency indicating that binding has occurred. Depending on the size of the drug, the change might be big or small. If too small to be detected, drug can be additionally labelled. The activity of the protease after drug binding needs to be tested by addition of proteins and measurement of the degradation fragments.

Controls: addition of different drugs, addition of no drug, measurement of activity prior and after the binding to ensure that the protease is not deactivated during immobilisation onto the transducer.

8.10 ∆ z = 9.4 mm 8.11 implantable, injectable or circulating nanosensors. Challenges are biocompatibility, immunogenic response, sensitivity, robustness in terms of continuous response and monitoring.

Chapter 9

9.1 By protecting the drug, increasing the amount of locally delivered drug and enabling targeted delivery.

9.2 Modification of nanocarriers with molecules such as PEG, which prevent binding of opsonin to the surface of nanocarriers and macrophage uptake.

9.3 Encapsulation within the core of the nanocarrier and surface immobilisation.

9.4 a) V of spherical liposome = 5.2x10-25 m3 Mass of the drug (taking into account the density and the volume of the liposome) = = 6.5x10-22 kg We would need 500 quadrillion (500x1015) liposomes to encapsulate 1 pill ( typically 325 mg) b) within the hydrophobic bilayer c) incorporation of pH sensitive linker, which cleaves when pH changes; use of light to heat up and open liposomes (they need to be modified with appropriate group or nanoparticle), incorporation of enzyme sensitive molecules, which are degraded in the cell and lead to drug release.

9.5 Gold nanoparticles can be used both for biosensing/detection due to their plasmons, and can be employed to induce hyperthermia by irradiation at the appropriate wavelength. Magnetic nanoparticles can be used for magnetic field induced heating and MRI detection. Any material, which can act both as a diagnostic tool and therapeutic agent can be considered theranostic.

9.6. Most likely clathrin-dependent endocytosis 9.7. Hydrogels can be made of biocompatible polymers, additionally modified with useful functional groups and can absorb large amounts of water. Their mechanical properties can be tuned to reflect those of particular tissues.

Chitosan can be modified with silver nanoparticles using amide coupling between free chitosan amines and carboxylic acid modified silver nanoparticles, or using additional modifications and employ one of the biofunctionalization strategies discussed in Chapter 4. Appropriate functional groups (maleimide, tetrazole) can be added to chitosan through amine group.

9.8 a) Covalent: amide coupling between carboxylic group within folic acid and amine coated Ag@MNPs. Non-covalent: Coating Ag@MNP with cationic species to enable electrostatic attachment of negatively charged folic acid. b) AFM, cryo TEM, fluorescent microscopy if nanoparticles are additionally labelled with a fluorophore c) Folate receptor (FR)-specific antibody and folate receptor (FR) binding aptamer.

For possible biofunctionalisation routes consult Chapter 4. d) Use of other cell lines not containing folic acid receptor, use of naked (without folic acid) nanoparticles, maybe adding an agent to block a folic acid receptor prior to nanoparticle binding and checking if any particle bound non-specifically.

9.9 Theranostic magnetic nanoparticles can be used as contrast agents for MRI detection, and can heat up under alternating magnetic field causing the damage of the biological material in their presence. Magnetic nanoparticles, as well as many other non-degradable classes usually end up in liver and/or are excreted through kidneys.

9.10 a) Surface can be coated with reactive oxygen species (ROS)-producing nanomaterials such as TiO2 or Cu nanoparticles. The added value of semiconducting nanomaterials is that they can usually be activated by light.

Alternatively, nanomaterials that can release ions, which might interfere with bacterial biochemical pathways might be used as well, such as Ag nanoparticles. b) Advantages: usually rely on production of ROS species, which can interfere with multiple biochemical pathways and do not lead to development of resistance.

Disadvantages: production and stability of nanomaterials, scale up, potential toxicity and environmental impact.

9.11. a) By modifying them with species such as PEG molecules which prevent opsonisation and macrophage recognition b) Cryo TEM, fluorescence microscopy if nanoparticles are additionally modified with fluorophore c) Intravenous injection: gets directly to the blood stream, prolonged circulation needed, danger of aggregation. Barriers: enzymes and macrophages in blood stream, penetration to the solid tissue (tumour stroma), cell membrane, enzymatic degradation within the cell.

Oral delivery: overcoming low stomach pH in addition to other barriers; macrophages, stroma, cell membrane, enzymes out of and in the cell.

9.12. Check Research Report 9.6