Nanotechnology and Life Sciences Copenhagen – October 2003 Mathias Brust Centre for Nanoscale Science, Department of Chemistry The University of Liverpool

THE USE OF BIOMOLECULAR TOOLS FOR NANOSTRUCTURE MANIPULATION THE USE OF NANOSTRUCTURES AS TOOLS IN BIOMOLECULAR AND BIOMEDICAL SCIENCES DRUG DELIVERY GENE DELIVERY DNA DETECTION BIO-ARRAYSBIO-MARKERS NANO-WIRES SENSORS MOLECULAR ELECTRONICSPHOTONICS NANOSTRUCTURE SELF-ASSEMBLY CATALYSIS MOLECULAR RECOGNITION ACTUATORS LIGHT SCATTERING PROBES CONTROLLED RELEASE FLUORESCENT Q-DOTS INFORMATION STORAGE SCANNING PROBE MICROSCOPY

Figure 2. A gap currently exists in the engineering of small-scale devices. Whereas conventional top-down processes hardly allow the production of structures smaller than about nm, the limits of regular bottom-up processes are in the range of about 2-5 nm. As a result of their own dimensions, two different types of compounds appear to be suited for addressing that gap: 1) biomolecular components, such as proteins and nucleic acids, and 2) colloidal nanoparticles comprised of metal and semiconductor materials.

DNA as building material for nanostructures N. Seeman et al.

DNA as template for growing Ag microwires between Au microelectrodes. Braun et. al., Nature 1998, 391,775

DNA-based assembly of gold nanostructures. Alivisatos et.al.,Angew.Chem.Int.Ed.,1999, 38,1808

DNA as a Linear Template for Electrostatically Controlled Assembly Protonation of aminodextran- stabilised gold colloids and electrostatic attachment to DNA  m 150 nm

Aminodextran MW: moles amine/mole Citrate-stabilised gold nanoparticles n Stabilisation of Gold Colloids by Polymer Capping

5`-Thiol-Modified oligonucleotides. 3`-Thiol-modified oligonucleotides. Attachment of DNA to gold nanoparticles via thiol- modified oligonucleotides 5’-Thiol modifier C6 S-S 3’-Thiol modifier C3 S-S CPG Au

Nanostructure assembly by DNA hybridisation

Control sample without complementarity 100 nm

Mirkin, C.A. et.al. Nature 1996, 382, 607. DNA/Au Nanostructures

Au-S-AAAAACGAGTGCTAAGGATCCGAA TTCACTGCAGATATCCATTCGAAAAA-S-Au GCTCACGATTCCTAGGCTTAAGTGACGTCTATAGGTAAGC 5`3` 5` 3` linker strand

Bioanalytical Applications

Colour based DNA detection and discrimination of mismatches

Figure 24. Scanometric detection of nucleic acids in DNA chip analyses. Capture oligonucleotides are immobilized on glass slides and used for the specific binding of target nucleic acids. Oligonucleotide- functionalized gold nanoparticles are employed as probes in solid-phase DNA hybridization detection. Subsequent to a silver enhancement step, the immobilization of the colloidal gold probe is detected by scanning the glass substrate with a conventional flat-bed scanner. The extraordinary sharp melting point of the immobilized DNA-nanoparticle networks (see melting curves on the right) allows single- mismatches to be detect by means of this method.

Figure 21. Sensing of DNA hybridization by means of SPR. A gold sensorchip functionalized with capture oligonucleotides S1 (A), is used to bind complementary target DNA S2 specifically (B). Introduction of a colloidal gold-oligonucleotide conjugate S3 allows the detection of the surface-immobilized complementary target (C). The corresponding SPR curves (reflectance as a function of the SPR angle) show the large increase in the transition from B to C, compared to that from A to B. Detection based on surface plasmon resonance (SPM)

Figure 20. Electrical detection of biorecognition processes. Receptor groups such as antibodies or DNA oligomers are immobilized in the gap between two microelectrodes and used as a capture agent to bind complementary target molecules specifically. In a sandwich-type assay, the captured analyte is tagged with colloidal gold by a second biological recognition unit. Subsequent reductive deposition of silver leads to the formation of a conducting metal layer which short-circuits the two electrodes. Electrical detection

Figure 19. Formation of a metal-Cytochrom c (Cc) metal sandwich for SERS. The heme-containing redox enzyme Cc adsorbs to citrate-stabilized gold particles in an oriented fashion through its lysine-rich heme pocket. The resulting conjugates are used to deliver the protein to colloidal Ag for SERS measurement. Surface Enhanced Raman Scattering (SERS)

Figure 1. Optical properties of PRPs. (a) A color photograph of three PRPs illuminated with white light. The particles were chosen so that their PR peak wavelengths would be red, green, and blue, respectively. The distance between the red and green particles is ~4 m. The weaker intensity of the blue PRP can be qualitatively seen by the overexposure of the film for the green and red images. (b) Spectral curves: the normalized intensity of scattered light as a function of wavelength for the three particles shown in (a). The two PRPs whose peak wavelengths are shifted by ~100 nm have ~10% overlap of intensity at the peak of their respective plasmon resonances. The full-width-at- half-height of the PRP with peak centred at ~440 nm is ~40 nm. (Figures (a) and (b) reproduced from [2] with permission. Copyright 2000 National Academy of Sciences, USA.)2 Detection based on resonance light scattering

Figure 2. Instrumentation for optical observation of PRPs. (a) Evanescent mode where the light is incident on a prism supporting an index-matched substrate at an angle resulting in TIR. Darkfield illumination introduced (b) via an optical fibre from the side, (c) using a standard, commercially available, darkfield condenser, and (d) using a brightfield/darkfield objective lens.

E-coli probed with gold Eub338. Identification of bacteria by selective binding to r-RNA

Figure 22. A) Fluorescence emission spectra of semiconductor quantum dots; B) cross-section through dual- labeled mouse fibroblasts. The actin fibers are stained red. The nonspecific labeling of the nuclear membrane by both the red and the green probes results in a yellow color. Fluorescence detection: Semiconductor Nanoparticles -single particle detection -single wavelength excitation -large gap between excitation and emission -same chemistry for all colours -no photobleaching

Figure 1: Detection of cancer marker Her2 with QD-IgG. (A, C) Fixed breast cancer SK-BR-3 cells were incubated with monoclonal anti-Her2 antibody and goat anti-mouse IgG conjugated to QDs. Her2 was clearly labeled with (A) QD 535–IgG and (C) QD 630–IgG (B, D). When cells were incubated with normal mouse IgG and QD-IgG, there were no detectable or very weak nonspecific signals on the cell surface. The nuclei were counterstained with Hoechst (blue). Filter sets ex nm/em nm and ex nm/em nm were used for QD 535 and QD 630, respectively. Scale bar, 10 m.

Figure 1 The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) and the preparation of serotonin-labeled nanocrystals (SNACs). S.J. Rosental et al. J. Am. Chem. Soc., 124 (17), , 2002.

Figure 6 Left: differential interference contrast images. Right: identical field of view; fluorescence images. Top: HEK cells with hSERTs in the cell membrane. Fluorescence labeling of the hSERTs in the membrane is clearly visible. Center: HEK cells that do not have hSERT in the cell membrane. No fluorescence labeling is visible. Right: HEK cells with hSERT in the membrane. These cells were exposed to an antagonist before being exposed to LSNACs. No fluorescence labeling is visible.

The images of four blocks inthe microarray before(left) and after silver enhance(right)

The images of the microarray before(left) and after silver enhance(right)

Fabrication of “bar-code” particles for multiplexing

Figure 1 Nanobarcodes particles (NBC). (A) Schematic image demonstrating range of possible dimensions and one possible striping pattern. (B) Optical microscope image of a single particle. The particle contains alternating sections of Ag and Au and is 6.3 m in length. Ag is the brighter material at this wavelength (405 nm). The apparent difference in the thickness of the Ag and Au stripes is due to the difference in brightness, not to a true difference in particle diameter.

Invasive intracellular tools For example: Gene gun technology The Future ? shoot at tissue