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) School of Biomedical and Molecular Sciences (SBMS) Postgraduate Medical School (PGMS) 5* RAE rating Leading optoelectronics / photonics group Queen’s Award 2002 for 20 year contribution Extensive Collaborations (Bookham Technology, Thales, IQE, Qinetiq, Infineon...) 5* RAE rating Leading early work on DNA probes for infectious diseases Extensive collaborations with pharmaceutical companies (GlaxoSmithKline, Pharmacia/Pfizer, Xenova, AstraZenecca, Oxagen, Cyclacel …) 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 Oncology Molecular toxicology Pharmacology Molecular Genetics Functional Genomics Quantum Dots Photonic Devices Ultrafast photonics Optical Spectroscopy School of Electronics & Physical Sciences School of Biomedical and Molecular Sciences Postgraduate Medical School

Relevant Activities at Surrey Oncology Molecular toxicology Pharmacology Molecular Genetics Functional Genomics Quantum Dots Photonic Devices Ultrafast photonics Optical Spectroscopy

Oncology Molecular toxicology Pharmacology Molecular Genetics Functional Genomics Quantum Dots Photonic Devices Ultrafast photonics Optical Spectroscopy BiosensorsComputational biophotonics Relevant Activities at Surrey

Co-applicants Physicists Biologists Clinician Helen ColeyOncology Fiona Green Functional Genomics George KassMolecular Toxicology Nick Plant Molecular Toxicology JohnJoe McFaddenMolecular Genetics Nick TomsPharmacology Jeremy AllamFemtosecond photonics Aleksey AndreevQuantum Dots David CareySpectroscopy/Microscopy Ortwin HessComputational Biophotonics Stephen SweeneyIntegrated biophotonic sensors Sub ReddyBiosensors

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...

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

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 Biophotonic Solutions

Molecular probes The SBMS Experience commercialisation – recent work

PCR-ELISA for diagnosis of meningococcal disease in blood substrate colour product This or similar test widely used in clinical laboratories around the world BUT takes hours (too slow! - patients may die of meningitis within hours of first symptoms) can only be performed in specialist labs Meningococcal DNA Patient sample PCR amplification DNA extraction enzyme ELISA Plate hospitals like these!! ELISA Plate Scanner Newcombe, … McFadden 1996 J.Clin.Microbiol. 34,

Quantum Dot ELISA-PCR for diagnosis of meningococcal disease in blood substrate colour product Compare with existing ELISA-PCR to benchmark quantum dot probes Meningococcal DNA Patient sample PCR amplification DNA extraction ELISA Plate hospitals like these!! ELISA Plate Scanner Quantum Dots

Quantum Dot diagnosis of meningococcal disease in blood single molecule QD Detection Meningococcal DNA Patient sample DNA extraction Quantum Dots direct Without PCR, the test should be much quicker and more easily applied in clinical labs

Quantum Dot multiplex detection of meningitis pathogens in blood single molecule QD Detection DNA Patient sample DNA extraction Quantum Dots direct 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 single molecule QD Detection DNA Patient sample DNA extraction Quantum Dots direct Genotyping, for diagnosis or for research, may employ tens or even hundreds of different DNA probes

Genetic Disease: Quantum Dots for Haplotyping or FRET ? Are genetic markers on the same or different chromosomes?

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 spatio-temporal imaging of neuron photonic readout of rotary biomolecular motors, protein folding, etc QD molecule biomolecular motor

PBG optical waveguide Nano-VCSEL Laser 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) Photonic Bandgap Biosensor

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 10 5 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 Medical Biology Photonics Infineon Thales Royal Surrey County Hospital GlaxoSmithKline Pharmacia/Pfizer Xenova plc AstraZenecca Oxagen Cyclacel St George’s Hospital Medical School Bookham Technology Qinetiq IQE

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 Ca 2+ 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 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.

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 10 0 nm to 10 2 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).

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 CW laser PMT scanner filter objective dichroic beamsplitter Fluorophore e.g. GFP, QD fluorescence excitationrapid decay Standard in molecular biology Confocal variant for 3D imaging

Advanced Microscopy / Cytometry Multiphoton Absorption Microscopy fs laser PMT scanner filter objective dichroic beamsplitter Fluorophore e.g. GFP, QD fluorescence 2-photon excitation rapid decay 3D imaging reduced UV-mediated cellular damage reduced photobleaching Denk et al, 1990

Advanced Microscopy / Cytometry Coherent / Nonlinear Microscopy: SHG fs laser PMT scanner filter objective dichroic beamsplitter Fluorophore e.g. GFP, QD h h SHG 2h No fluorophores needed probes  (2) reveals symmetry breaking (e.g. cell membranes)

Advanced Microscopy / Cytometry Coherent / Nonlinear Microscopy: THG fs laser PMT scanner filter objective dichroic beamsplitter Fluorophore e.g. GFP, QD h h THG 2h probes  (3) sensititive to refractive index h Barad et al, 1997

Advanced Microscopy / Cytometry Coherent Anti-Stokes Raman Scattering Microscopy dual fs laser PMT scanner filter objective dichroic beamsplitter h p 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 h S h p h AS |0 > |1 >

camera filter z excitation density <100nm TIRF Microscopy prism total internal reflection (TIR) of excitation beam elimination of background excitation light elimination of out of focus fluorescence objective -> individual fluorophore imaging at the cellular plasma membrane. prism excitation fluorescence

Develop multiphoton TIRF microscope PMT filter objective -> limit UV-mediated cellular damage and reduce photobleaching prism Combined TIRF- Multiphoton Microscopy & Laser Scanning Cytometry Apply developed TIRF technology to laser scanning cytometry -> selective high-resolution detection of perimembrane fluorescence scanner excitation 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 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. Select projects which:

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 commercialised PCR- based diagnostic test FIRST PCR test for meningitis