Functional Photonics for Single Bioentities An application for a Platform Grant in Biophotonics from the University of Surrey

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Functional Photonics for Single Bioentities An
application for a Platform Grant in Biophotonics
from the University of Surrey
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Present today Jeremy Allam Professor of
Ultrafast Optoelectronics Overview
Questions related to Photonics JohnJoe
McFadden Professor of Molecular
Genetics Questions related to biomedical
aspects David Carey EPSRC Advanced Research
Fellow Interdisciplinarity and nanotechnology
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The Surrey Scene
School of Electronics Physical Sciences (SEPS)
5 RAE rating Leading optoelectronics /
photonics group Queens Award 2002 for 20 year
contribution Extensive Collaborations (Bookham
Technology, Thales, IQE, Qinetiq, Infineon...)
School of Biomedical and Molecular Sciences
(SBMS)
5 RAE rating Leading early work on DNA probes
for infectious diseases Extensive collaborations
with pharmaceutical companies (GlaxoSmithKline,
Pharmacia/Pfizer, Xenova , AstraZenecca, Oxagen,
Cyclacel )
Postgraduate Medical School (PGMS)
Formed 2000 to support health-related research
Link to NHS and clinicians (St Georges Hospital
Medical School, Royal Surrey County Hospital)
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Relevant Activities at Surrey
School of Electronics Physical Sciences
School of Biomedical and Molecular Sciences
Postgraduate Medical School
Oncology
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Relevant Activities at Surrey
Oncology
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Relevant Activities at Surrey
Quantum Dots
Photonic Devices
Ultrafast photonics
Optical Spectroscopy
Molecular toxicology
Molecular Genetics
Functional Genomics
Pharmacology
Oncology
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Co-applicants
Physicists
Jeremy Allam Femtosecond photonics Aleksey
Andreev Quantum Dots David Carey Spectroscopy/Mi
croscopy Ortwin Hess Computational
Biophotonics Stephen Sweeney Integrated
biophotonic sensors
Sub Reddy Biosensors
Biologists
Fiona Green Functional Genomics George
Kass Molecular Toxicology Nick Plant Molecular
Toxicology JohnJoe McFadden Molecular
Genetics Nick Toms Pharmacology
Clinician
Helen Coley Oncology
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University Support for Relevant Interdisciplinary
Research

• Interdisciplinary Research Institutes
• Advanced Technology Institute
• 5M from JIF 5M from UniS
• incorporating photonics and electronics research
• extensive new device fabrication facilities
• centre of excellence in Medical Research
• opening March 2005
• to promote health related research and build
  University-NHS links
• Infrastructure funding
• Functional Genomics Laboratory
• 2.3M from SRIF1
• genomics and proteomics facilities
• Nano-bioelectronics facility
• 3.8M from SRIF2
• nanofabrication, e.g. focussed ion beam,
• surface plasmon resonance apparatus
• staff recruitment ...

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Biomedical Objectives

• Our proposal is strongly focussed on important
  biomedical applications
• Infectious disease diagnosis
• detection and identification of pathogens
• Pharmacology
• drug-receptor dynamics in health and disease
• Human genetics
• genotyping and haplotyping

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Molecular probesBiophotonic Solutions

• DNA-conjugated or antibody-conjugated quantum
  dots coupled to direct detection of signal for
  single molecule detection.
• Multiplex quantum dots for parallel probing.

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Molecular probesThe SBMS Experience
1990
1990
1992
2004 recent work
1987
1987
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PCR-ELISA for diagnosis of meningococcal disease
in blood
Patient sample
Newcombe, McFadden 1996 J.Clin.Microbiol. 34,
1637-1640
DNA extraction
Meningococcal DNA
PCR amplification
enzyme
ELISA Plate Scanner
colour product
substrate
ELISA Plate hospitals like these!!

• This or similar test widely used in clinical
  laboratories around the world
• BUT
• takes 24-36 hours
• (too slow! - patients may die of meningitis
  within hours of first symptoms)
• can only be performed in specialist labs

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Quantum Dot ELISA-PCR for diagnosis of
meningococcal disease in blood
Patient sample
DNA extraction
Meningococcal DNA
PCR amplification
Quantum Dots
ELISA Plate Scanner
colour product
substrate
ELISA Plate hospitals like these!!
Compare with existing ELISA-PCR to benchmark
quantum dot probes
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Quantum Dot diagnosis of meningococcal disease
in blood
Patient sample
DNA extraction
Meningococcal DNA
direct
Quantum Dots
single molecule QD Detection
Without PCR, the test should be much quicker and
more easily applied in clinical labs
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Quantum Dot multiplex detection of meningitis
pathogens in blood
Patient sample
DNA extraction
DNA
direct
Quantum Dots
single molecule QD Detection
Rapid identification of specific agent involved
(there are many that cause meningitis), or
detection of drug-resistance gene, may be vital
for implementing appropriate treatment regime
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Genetic Disease Quantum dots for multiplex SNP
genotyping
Patient sample
DNA extraction
DNA
direct
Quantum Dots
single molecule QD Detection
Genotyping, for diagnosis or for research, may
employ tens or even hundreds of different DNA
probes
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Genetic Disease Quantum Dots for Haplotyping
Are genetic markers on the same or different
chromosomes?
or
?
FRET
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Functional QD Probes

• optical properties of QDs depend on electric
  field, molecular vibrations, orientation,
  proximity, etc, hence QDs as functional probes
• real-time spatio-temporal dynamics of
  biomolecular function
• We will calculate QD properties and hence design
  functional probes. Information will be supplied
  to collaborators for fabrication of the QDs

photonic readout of rotary biomolecular motors,
protein folding, etc
spatio-temporal imaging of neuron
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Integrated Biophotonic Sensors

• Alternative approaches to high-sensitivity,
  multiplexed biophotonic sensors
• resonance condition for high sensitivity (e.g.
  dual-stripe mode-locked laser)
• spatial readout
• exploit bio-nano size match
• new operational modes e.g. photonic bandgaps
  (PBG)

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What it will mean for us
exploit existing research strengths in new
directions fully exploit strong investment in
infrastructure and capital equipment retain
flexibility in staffing and training make an
impact in an important emerging research field
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ContentsPersonnelEnvironmentInfrastructure
fundingStrategy Biomedical ObjectivesSpecific
Projects Nanoparticle and Quantum Dot Molecular
Probes Functional Photonic Probes Advanced
Microscopy / Cytometry Integrated Biophotonic
Sensors Computational biophotonics
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Genetic Disease Quantum dot-ELISA for
haplotyping (to determine whether genetic markers
are on the same or different chromosomes)
or
?
FRET
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Molecular probescurrent limitations

• Direct DNA probing is limited by stoichiometry of
  DNA hybridisation one target binds one
  probe-signal molecule.
• Signal detection is relatively insensitive need
  about 105 signal molecules for detection.
• Current DNA probe applications overcome this
  problem by employing polymerase chain reaction
  (PCR).
• PCR amplifies target DNA molecules more than one
  million fold. Amplified PCR product can then be
  detected by conventional DNA probes.
• But.
• This makes DNA probe tests lengthy, expensive,
  requiring specialist laboratories and trained
  personal, and prone to errors particularly from
  PCR contamination.
• DNA probes tests generally utilise the same (or
  up to 4 different) output signal(s) so multiple
  tests are usually performed serially.

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Quantum Dot Biomolecular Probes

• Semiconductor Quantum Dots (QDs) (diameters of
  1 - 5nm)
• Increased brightness and lifetime
• decreased spectral width (size selection) -gt
  higher multiplexing
• smaller size -gtreduced steric hindrance
• commercially-available, bioconjugation
  well-established
• existing DNA and antibody probe systems developed
  at Surrey will be modified to incorporate QD
  probe readout
• limits of quantum dot multiplexing will be
  studied for applications in e.g. SNP genotyping.
• investigate new ways to control size, shape and
  location of QD by electrochemical synthesis
  within polymer film micropores
• QDs combined with TIRF to study cell surface
  events
• proximity effects studied for variants of FRET
  (e.g. for haplotyping)

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Strategy
A Platform to underpin a new research direction
in biophotonics (not a responsive mode minus
consumables) Address staff continuity, training,
fast-start-up of research Flexible baseline
funding Strong support from University .
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Related Grant Applications
MRC Capacity Building Area Studentships
"Intracellular imaging/dynamics" 2 PhD
studentships EPSRC Application Nanoelectronic
Circuits in silicon-on-insulator Sweeney and
Reed integration of optoelectronics with Si
Platform applications including biosensing EU
Framework 6 Application Gallium Nitride Epitaxy
and Devices for New Applications FP6 (Thales)
Sweeney, Sale, Adams, Hosea integration of
wide-gap light-emitting diodes with passive
waveguides and microfluidics, and applications
including biosensing Nanobio-electronics Mendoza
- nanotubes for EEGs sleep Sleep research centre
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External Collaborations
Photonics

Biology

Medical
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Infectious Disease Diagnosis

• Limitation of Current Technologies
• Direct DNA antibody probes are highly specific
  but relatively insensitive
• DNA amplification using PCR increases
  sensitivity, but needs (gt50), time (several
  days), and expertise
• Need for Improved Solutions
• Time can be critical in clinical situations...
  e.g. during assays or to prevent disease
  progression
• numerous topical examples where current tests
  inadequate (SARS, chemical / biological weapons
  agents multidrug resistant bacteria).
• Objective
• develop biomolecular probes based on biophotonics
  that are specific, sensitive (single virus or
  bacteria), fast, and low cost
• A new generation of molecular diagnostic tools is
  urgently needed that are fast, relatively
  inexpensive and may be applied at the bedside or
  the GPs surgery. It is the enabling technology
  for these new solutions which we are addressing
  in our research.

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Pharmacology

• Drug-receptor dynamics in health
• intracellular signalling triggered by specialised
  regions in plasma membrane, exhibits dynamics on
  sub-millisecond timescale
• Imaging intracellular Ca2 Dynamics
• Individual Receptor Trafficking Fluorophore
  (e.g. GFP)-tagged receptor
• Death Receptors Receptor-receptor and other
  interactions under stress conditions
• Drug-receptor dynamics in disease
• modern cancer therapeutics are directed at growth
  factor receptors and signal transduction pathways
  
• Assays for patients treated with these agents
  will be established using patient biopsy material
  obtained from the St Lukes Cancer Centre.
• We will further develop membrane-localised
  microscopy methods and apply them to the study of
  drug-receptor interactions, and their consequence
  for health and disease.

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Human Genetics

• High Throughput Genotyping
• Single Nucleotide Polymorphism detection used to
  identify susceptibility for common diseases e.g.
  heart disease, cancer
• Multiple (e.g. 20) SNP probes needed to identify
  phenotypes
• Serial processing is time-consuming
• Highly-muliplexed SNP probes based on QD tags
  will allow high-throughput screening.
• Haplotype determination
• location of disease-associated genetic variation
  on the chromosome inherited from mother or father
  ?
• Usually takes inheritance studies over three
  generations
• Biophotonics approaches to haplotyping...
• We aim to develop high-speed cost effective
  genotyping techniques for use in clinical /
  counselling environments.

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Human Genetics

• Haplotype determination
• location of disease-associated genetic variation
  on the chromosome inherited from mother or father
  ?
• Usually takes inheritance studies over three
  generations
• Biophotonics approaches to haplotyping...
• We aim to develop high-speed cost effective
  genotyping techniques for use in clinical /
  counselling environments.

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Nanoparticle Biomolecular Probes
Metallic nanoparticles (NP) (from 100nm to 102
nm) can be used for non-fluorescent labels.
Interaction with probe light is through light
scattering, plasmon resonance or local
enhancement of nonlinear optical response. We
will functionalise a number of NP configurations
(e.g. dots, shells, rods, ...) with the aim of
optimising the sensitivity or specificity for
different detection mechanisms including
non-linear microscopy (TM, SR, JA). This activity
will be supported by theoretical calculations of
the response of nanoparticles to driving fields
in model biological environments (ADA, OH).
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Advanced Microscopy / Cytometry

• Total Internal Reflection Fluorescence (TIRF)
  Microscopy
• Individual fluorophore imaging at the cellular
  plasma membrane.
• Develop multiphoton TIRF microscope (limit
  UV-mediated cellular damage and reduce
  photobleaching).
• Laser Scanning Cytometry (LSC)
• Apply developed TIRF technology to LSC to enable
  selective high-resolution detection of
  perimembrane fluorescence.
• Coherent Nonlinear Microscopy
• No fluorophore required.
• Second harmonic generation reveals
  symmetry-breaking (e.g. at cell membranes).
• Third harmonic generation gives structural
  information.
• Coherent Anti-Stokes Raman Scattering is
  sensitive to molecular vibrations.
• Very promising for chemically-selective
  label-free dynamic microscopy in biomedical
  science.
• Multiphoton and multiharmonic microscopy will be
  integrated into a single nonlinear microscope.

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Advanced Microscopy / Cytometry
Conventional Laser Scanning (epifluorescence)
Microscope
PMT
filter
scanner
dichroic beamsplitter
CW laser
objective

• Standard in molecular biology
• Confocal variant for 3D imaging

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Advanced Microscopy / Cytometry
Advanced Microscopy / Cytometry
Multiphoton Absorption Microscopy
PMT
rapid decay
filter
fluorescence
2-photon excitation
scanner
dichroic beamsplitter
fs laser
Fluorophore e.g. GFP, QD
objective

• 3D imaging
• reduced UV-mediated cellular damage
• reduced photobleaching

Denk et al, 1990
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Advanced Microscopy / Cytometry
Advanced Microscopy / Cytometry
Coherent / Nonlinear Microscopy SHG
PMT
filter
hn
SHG
2hn
scanner
dichroic beamsplitter
hn
fs laser
Fluorophore e.g. GFP, QD
objective

• No fluorophores needed
• probes c (2)
• reveals symmetry breaking (e.g. cell membranes)

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Advanced Microscopy / Cytometry
Advanced Microscopy / Cytometry
Coherent / Nonlinear Microscopy THG
PMT
hn
filter
THG
hn
2hn
scanner
dichroic beamsplitter
hn
fs laser
Fluorophore e.g. GFP, QD
objective

• probes c (3)
• sensititive to refractive index

Barad et al, 1997
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Advanced Microscopy / Cytometry
Advanced Microscopy / Cytometry
Coherent Anti-Stokes Raman Scattering Microscopy
PMT
filter
hnp
hnAS
hnS
hnp
scanner
dichroic beamsplitter
1 gt
0 gt
dual fs laser
objective

• requires dual-wavelength fs laser
• resonant with vibrational energies
• sensitive to chemical composition
• Very promising for chemically-selective
  label-free dynamic microscopy in biomedical
  science.

Zambusch et al, 1999
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TIRF Microscopy
camera

• total internal reflection (TIR) of excitation
  beam
• elimination of background excitation light
• elimination of out of focus fluorescence

filter
fluorescence
-gt individual fluorophore imaging at the cellular
plasma membrane.
objective
z
lt100nm
prism
excitation
excitation density
prism
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Combined TIRF-Multiphoton Microscopy Laser
Scanning Cytometry
PMT
filter

• Develop multiphoton TIRF microscope

-gt limit UV-mediated cellular damage and reduce
photobleaching
fluorescence

• Apply developed TIRF technology to laser scanning
  cytometry
• -gt selective high-resolution detection of
  perimembrane fluorescence

objective
prism
excitation
scanner
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Objectives of the proposal

• identify important biomedical problems with
  potential photonic solutions
• implement state-of-the-art photonic solutions for
  routine use by biologists
• develop new biophotonic methods

Select projects which

• play to strengths in photonics and biology
• involve activity on both bio- and -photonics
  aspects, to rapidly build collaborations
• make a specific contribution to the research
  field, rather than catch up with advances
  elsewhere
• exploit our new facilities in nano-fabrication,
  ultrafast lasers, advanced simulation, functional
  genomics, etc
• are synergistic with emerging research directions
  at Surrey such as nano-bio-electronics, etc
• have an identified user or customer for any
  technology being developed
• are benchmarked, e.g. biosensors will be compared
  to state-of-the-art detection systems developed
  at UniS.

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Underlying technology - DNA probes
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Computational Biophotonics

• A strong theoretical programme underpins the
  experimental activity
• Quantum dot (QD) calculations
• design of QDs and QD molecules for functional
  probes
• Simulation of advanced photonic structures
• photonic bandgaps
• multi-section lasers
• Related activities
• simulation of biomolecular motors
• bioinformatics

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Biophotonics is the science of generating and
harnessing light (photons) to image, detect and
manipulate biological materials.Biophotonics is
used in BIOLOGY to probe for molecular
mechanisms, function and structure. It is used in
MEDICINE to study tissue and blood at the macro
(large-scale) and micro (very small scale)
organism level to detect, diagnose and treat
diseases in a way that are non-invasive to the
FIRST commercialised PCR-based diagnostic
test FIRST PCR test for meningitis