Biosensors for efficient capture of biological information Current technology relies on inefficient systems for capture of biological information: –Information encoded in the DNA/RNA of microbes for infectious disease diagnosis –Information encoded in chemical structures of metabolites/drugs in clinical laboratory diagnosis of metabolic disease.

DIG-ELISA PCR for diagnosis of meningococcal meningitis substrate product SIGNAL Newcombe, J., et al J.Clin.Microbiol. 34, Meningococcal DNA enzyme Patient sample PCR amplification DNA extraction

Nano-bioelectronic Devices to efficiently capture biological signals target capture signal transfer signal transduction signal capture

DNA chip biosensor (Cao et al, Science, 2002) DNA signal  light signal Chip with bound capture probe – captures target DNA Target DNA (bound to chip) captures gold nanoparticles labelled with Ramen dye Laser bean  Raman scattering  signal Can detect 20 femtomoles – similar to PCR

TRANSCRIPTOMICS

Green Fluorescent protein to track cells and examine gene expression

Flow Cytometry SBLS has FACSCAN (Becton Dickenson) and FACSCalibur (BeckmanCoulter) Flow cytometry widely used to measure fluorescent intensity and proportion of target cells in cell population, particularly immune cells

Quantum Dots fluoresce with a narrow and symmetric emission spectrum that depends directly on the size of the crystal. can be fine-tuned to emit light at a variety of wavelengths simply by altering the size of the core constitute a set of multicoloured molecular beacons (up to 40,000 colours) for use in imaging Soluble quantum dots injected into frog embryos – only distributed to the offspring of the injected parent cell, and did not diffuse out of the cell used to track cell lineages. Can be tagged with antibody or DNA probes

Possible use of quantum dots? DNA fingerprinting with multiple allele-specific probes Widely used in forensic science, genetic typing and infectious disease diagnosis

2 Photon Microscopy In confocal microscopy the exciting laser must be very bright to allow an adequate signal-to-noise ratio. –photobleaching –Phototoxicity In 2 photon microscopy the signal is generated by two lower-energy (infrared) photons that are absorbed contemporaneously (within 1 femtosecond). –More focussed beam –Less toxicity

Biophotonics in SBLS Dr George Kass (Toxicology) Dr Nick Toms (Pharmacology) Professor Ian Kitchen (Pharmacology) Dr Lesley-Jane Reynolds (Microbiology) 1.Confocal Microscopy (Zeiss 510 META) Live cell (e.g. FRET) & fixed cell imaging (e.g. immunofluorescence) 2. Epifluorescence Microscopy Single cell live dynamic fluorescence (e.g. intracellular Ca 2+ imaging) 3. Flow Cytometry Cell population analysis of cellular fluorescence 4. Quantitative Autoradiography Radioligand (e.g. [ 3 H]drug) binding to tissue sections

Low Green Fluorescence High Red Fluorescence Excitation (488 nm) FRET eYFP Protein dsRed Protein Fluorescence Resonance Energy Transfer (FRET) Amino acid chain (Caspase-3 Target) Healthy NeuronesDying Neurones Excitation (488 nm) FRET X No Red Fluorescence High Green Fluorescence Death Enzyme “Caspase-3”

Green Fluorescence Red Fluorescence Excitation (488 nm) FRET eYFP Protein dsRed Protein Healthy Neurones

A Dying Neurone Excitation (488 nm) FRET X No Red Fluorescence High Green Fluorescence Death Enzyme “Caspase-3”

Confocal Analysis of Mitochondria

Immunofluorescence labelling of a Myelinating Brain Cell (Oligodendrocyte) Confocal Z-stack panoramic movie (click on image to run)

Quantitative Autoradiography fmol/mg NSB Binding of a [ 3 H]drug to glutamate receptors in rat brain.