Functional Photonics
for Single Bioentities
An application for a
Platform Grant in Biophotonics
from the
University of Surrey
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
The Surrey Scene
School of Electronics & Physical Sciences (SEPS)
5* RAE rating
Leading optoelectronics / photonics group
Queen’s 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 George’s Hospital Medical School, Royal Surrey
County Hospital)
Relevant Activities
at Surrey
School of Electronics & Physical Sciences
Quantum
Dots
Ultrafast
photonics
Photonic
Devices
Optical
Spectroscopy
School of Biomedical and Molecular Sciences
Molecular
Genetics
Functional
Genomics
Pharmacology
Molecular
toxicology
Postgraduate Medical School
Oncology
Relevant Activities
at Surrey
Quantum
Dots
Ultrafast
photonics
Molecular
Genetics
Functional
Genomics
Photonic
Devices
Optical
Spectroscopy
Pharmacology
Molecular
toxicology
Oncology
Relevant Activities
at Surrey
Quantum
Dots
Ultrafast
photonics
Photonic
Devices
Computational biophotonics
Molecular
Genetics
Functional
Genomics
Optical
Spectroscopy
Biosensors
Pharmacology
Molecular
toxicology
Oncology
Co-applicants
Physicists
Jeremy Allam
Aleksey Andreev
David Carey
Ortwin Hess
Stephen Sweeney
Femtosecond photonics
Quantum Dots
Spectroscopy/Microscopy
Computational Biophotonics
Integrated biophotonic sensors
Sub Reddy
Biologists
Fiona Green
George Kass
Nick Plant
JohnJoe McFadden
Nick Toms
Functional Genomics
Molecular Toxicology
Molecular Toxicology
Molecular Genetics
Pharmacology
Clinician
Helen Coley
Oncology
Biosensors
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
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
Molecular probes
Biophotonic Solutions
• DNA-conjugated or
antibody-conjugated
quantum dots coupled
to direct detection of
signal for single
molecule detection.
• Multiplex quantum
dots for parallel
probing.
Molecular probes
The SBMS Experience
1990
1990
commercialisation
1992
1987
1987
2004 – recent
work
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
colour
product
substrate
ELISA
Plate
Scanner
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
Quantum Dot ELISA-PCR for
diagnosis of meningococcal
disease in blood
Patient sample
DNA extraction
Meningococcal DNA
PCR amplification
Quantum Dots
colour
product
substrate
ELISA
Plate
Scanner
ELISA Plate
hospitals like these!!
Compare with existing ELISA-PCR to benchmark quantum
dot probes
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
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
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
Genetic Disease: Quantum
Dots for Haplotyping
Are genetic markers on the same or different chromosomes?
or
FRET
?
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
QD
molecule
biomolecular
motor
photonic readout of rotary biomolecular
motors, protein folding, etc
spatio-temporal imaging of neuron
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)
Nano-VCSEL Laser
Photonic Bandgap Biosensor
PBG optical
waveguide
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
Contents:
Personnel
Environment
Infrastructure funding
Strategy
Biomedical Objectives
Specific Projects:
Nanoparticle and Quantum Dot Molecular Probes
Functional Photonic Probes
Advanced Microscopy / Cytometry
Integrated Biophotonic Sensors
Computational biophotonics
Genetic Disease
Quantum dot-ELISA for
haplotyping
(to determine whether genetic
markers are on the same or
different chromosomes)
or
?
FRET
Molecular probes
current 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.
Quantum Dot
Biomolecular Probes
Semiconductor Quantum Dots (QDs) (diameters of ~ 1 - 5nm)
•
•
•
•
Increased brightness and lifetime
decreased spectral width (size selection) -> higher multiplexing
smaller size ->reduced 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)
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 ….
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
External
Collaborations
Infineon
Photonics
Thales
Bookham Technology
Qinetiq
IQE
GlaxoSmithKline
Pharmacia/Pfizer
AstraZenecca
Biology
Xenova plc
Cyclacel
Royal Surrey
County Hospital
Oxagen
St George’s Hospital
Medical
Medical School
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 $$ (>$50),
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
GP’s surgery. It is the enabling technology for these new solutions which
we are addressing in our research.
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 Luke’s 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.
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 highthroughput 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.
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.
Nanoparticle
Biomolecular Probes
Metallic nanoparticles (NP) (from 100nm to 102 nm) can be used for nonfluorescent 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).
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.
Advanced Microscopy
/ Cytometry
Conventional Laser Scanning (epifluorescence) Microscope
rapid
decay
PMT
filter
fluorescence
scanner
excitation
Fluorophore
e.g. GFP, QD
CW
laser
• Standard in molecular biology
• Confocal variant for 3D imaging
dichroic
beamsplitter
objective
Advanced Microscopy
/ Cytometry
Multiphoton Absorption Microscopy
rapid
decay
2-photon
excitation
PMT
filter
fluorescence
Fluorophore
e.g. GFP, QD
scanner
fs
laser
• 3D imaging
• reduced UV-mediated cellular damage
• reduced photobleaching
dichroic
beamsplitter
objective
Denk et al, 1990
Advanced Microscopy
/ Cytometry
Coherent / Nonlinear Microscopy: SHG
PMT
filter
hn
hn
SHG
2hn
Fluorophore
e.g. GFP, QD
scanner
fs
laser
dichroic
beamsplitter
objective
• No fluorophores needed
• probes c (2)
• reveals symmetry breaking (e.g. cell membranes)
Advanced Microscopy
/ Cytometry
Coherent / Nonlinear Microscopy: THG
PMT
hn
hn
filter
THG
2hn
scanner
hn
Fluorophore
e.g. GFP, QD
fs
laser
• probes c (3)
• sensititive to refractive index
dichroic
beamsplitter
objective
Barad et al, 1997
Advanced Microscopy
/ Cytometry
Coherent Anti-Stokes Raman Scattering Microscopy
PMT
filter
hnp
hnS hnp
hnAS
scanner
|1 >
|0 >
dual fs
laser
dichroic
beamsplitter
objective
• requires dual-wavelength fs laser
• resonant with vibrational energies
• sensitive to chemical composition
Very promising for chemically-selective label-free
Zambusch et al, 1999
dynamic microscopy in biomedical science.
TIRF Microscopy
filter
• total internal reflection (TIR) of excitation
beam
• elimination of background excitation light
• elimination of out of focus fluorescence
camera
fluorescence
-> individual
fluorophore
imaging at the cellular
plasma membrane.
objective
z
<100nm
prism
excitation
prism
excitation density
Combined TIRFMultiphoton Microscopy &
Laser Scanning Cytometry
PMT
filter
-> limit UV-mediated cellular damage
and reduce photobleaching
fluorescence
• Apply developed TIRF technology to
laser scanning cytometry
objective
excitation
scanner
• Develop multiphoton TIRF
microscope
prism
-> selective high-resolution detection of
perimembrane fluorescence
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-bioelectronics, 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.
Underlying technology DNA probes
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
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