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, 1637-1640.

Meningococcal DNA

enzyme

Patient sample

PCR amplification

DNA extraction

Nano-bioelectronic Devicesto efficiently capture biological signals

targetcapture

signaltransfer

signaltransduction

signalcapture

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 MicroscopySingle cell live dynamic fluorescence (e.g. intracellular Ca2+ imaging)

3. Flow CytometryCell population analysis of cellular fluorescence

4. Quantitative AutoradiographyRadioligand (e.g. [3H]drug) binding to tissue sections

Low Green Fluorescence High Red

Fluorescence

Excitation(488 nm) FRET

eYFPProtein

dsRedProtein

Fluorescence Resonance Energy Transfer (FRET)

Amino acid chain(Caspase-3 Target)

Healthy Neurones Dying Neurones

Excitation(488 nm)

FRETXNo Red

Fluorescence High Green Fluorescence

Death Enzyme“Caspase-3”

Green Fluorescence

Red Fluorescence

Excitation(488 nm)

FRETeYFP

ProteindsRedProtein

Healthy Neurones

A Dying Neurone

Excitation(488 nm)

FRETXNo 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 [3H]drug to glutamate receptors in rat brain.