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Day 1 Day 2
Day 1 - Tuesday, May 5, 2015
7:00 Check In & Registration
7:55 Welcome & Opening Remarks
Clinical Applications of Bioanalytical Sensors
Moderator: Oliver Plettenburg, Sanofi
8:00 Clinical Raman Spectroscopy
Juergen Popp
Professor and Chair, Institute of Physical Chemistry and Abbe Center of Photonics
Director, Leibniz Institute of Photonic Technology
Friedrich Schiller University
Understanding the cause of diseases, early disease recognition, targeted disease treatment and monitoring of treatment success are the underlying principles of the vision associated with modern biophotonics. Spectroscopic methods play a key role in turning this ambitious vision into reality because they allow for monitoring life processes on a molecular level. In this context Raman spectroscopy based approaches are extremely promising since they provide direct molecular contrast in a label free manner. Within this contribution, we describe some of our latest results concerning the application of Raman approaches for clinical diagnosis. We will start with highlighting the potential of Raman microspectroscopy for an early diagnosis and therapy of sepsis. In the field of sepsis, the fast identification of pathogens, their resistances and the specific host is crucial for choosing the appropriate initial antibiotic therapy. It will be shown that Raman holds great promise as point-of-care approach to address these challenging tasks. Furthermore, Raman provides a sensitive and selective diagnostic tool for cell and tissue analysis for an early diagnosis of diseases like e.g. cancer. Moreover, specially designed Raman fiber probes enabling optical biopsies during endoscopy are introduced. However, the low Raman scattering cross section often results in long acquisition times which can be reduced by utilizing non-linear Raman approaches like CARS (coherent anti-Stokes Raman scattering). CARS microscopy allows recording Raman images of single characteristic Raman bands in real time. We will present the development of a compact CARS microscope in combination with novel fiber laser sources for use in clinics.
8:40 Insight into Alzheimer’s Disease Precursors from Spectroscopy
Kerensa Broersen
Nanobiophysics Group
University of Twente
One of the most prominent hallmarks detected in the brains of patients suffering from Alzheimer’s disease is the deposition of amyloidogenic plaques. These plaques are largely composed of the amyloid-beta peptide. It has been demonstrated that, even though these plaques comprise an important and recurring feature for disease, that precursor forms of these plaques, called ‘oligomers’ or ‘protofibrils’ more potently affect neuronal functioning. One of the causes of early onset familial-associated Alzheimer’s disease (FAD) are mutations of the amyloid-beta peptide or mutations in the amyloid protein precursor processing enzymes. The mechanism by which hence formed variants of the amyloid-beta peptide cause disease still remains elusive. In aim to determine how amyloid beta peptide manifests its pathological effects, we monitored oligomer formation and aggregation using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy and immunohistochemistry. The resulting fibrils were structurally characterized with X-ray fiber diffraction, limited proteolysis and hydrogen/deuterium exchange monitored by FTIR and mass spectrometry. Differences could be observed in secondary structural organization, response to potential therapeutics and neuronal functioning. We demonstrate that a combination of spectroscopic, biochemical and cell biological techniques can serve as sensors in clarifying the potential underlying pathologic molecular pathways.

Key benefits:
• A combination of spectroscopic, biochemical and cell biological techniques can serve as ‘sensing device’ to investigate disease mechanism.
• Molecular insight into one of the most prominent neurodegenerative disorders.
• Potential for development of high-throughput therapeutic screening platforms.
• Identification of therapeutic targets.
9:05 Terbium-to-Quantum Dot FRET for Sensitive and Multiplexed Optical Biosensing
Niko Hildebrandt
Professor, NanoBioPhototonics
Université Paris-Sud
Applications based on Förster Resonance Energy Transfer (FRET) play an important role for the determination of concentrations and distances within nanometer-scale systems in vitro and in vivo in many fields of the life sciences. Using time-resolved luminescence spectroscopy and microscopy for the analysis of FRET systems offers several advantages concerning sensitivity and specificity. Two extraordinary materials concerning time-resolved FRET are luminescent terbium complexes (Tb) and semiconductor quantum dots (QDs). Both are frequently used as FRET donors in combination with organic dyes, fluorescent proteins and other fluorophores. However, their combination as Tb-QD donor-acceptor pair gives access to unique photophysical properties, which make this FRET-pair a valuable tool for highly sensitive multiplexed biosensing.

The presentation will start with an introduction to time-resolved FRET, terbium complexes and quantum dots, and their possible applications for the determination of subnanomolar concentrations and subnanometer distances. Then it will focus on recent results concerning FRET for ultrasensitive multiplexed biosensing, including homogeneous immunoassays, microRNA detection, enzyme kinetics, and crossing of the blood-brain barrier.

Benefits of talk:
• Introduction to FRET-based biosensing
• Multiplexing capabilities of quantum dot-based FRET
• High sensitivity homogeneous optical diagnostics
• Time-gated detection for ultra-low background signals
9:30 Raman Spectroscopy to Study Endothelial Dysfunction
Malgorzata Baranska
Faculty of Chemistry & Jagiellonian Center for Experimental Therapeutics (JCET)
Jagiellonian University
Clinical applications of Raman spectroscopy used to study in vitro endothelial cells, and ex vivo tissues of various mice models, i.e. atherosclerosis, diabetes, liver diseases are presented. Spectroscopic measurements were combined with AFM, SNOM, IR spectroscopy and/or fluorescence microscopy.

Endothelium plays an important role in cardiovascular system and it regulates vascular homeostasis. Confocal Raman imaging was used to monitor a molecular composition occurring in a single live human aorta endothelial cell (HaoEC) as a result of its uptake of fatty acids (e.g. arachidonic acid, AA). After a long-term incubation with AA the lipid droplets (LDs) are observed in the cell. The lipid droplet plays a role in diverse cellular functions that may be altered in metabolic syndromes, obesity, steatosis and atherosclerosis.

The other diseases, caused by the malfunction of endothelium is diabetes. In this work the en face aorta of healthy (C57) and diabetic mice (db/db model) was investigated in order to find spectroscopic and microscopic (AFM) differences.

Liver were taken from mice models fed on special diets, Low Carbohydrate High Protein (LCHP) or High Fat diet (HFD). LCHP model did not represent large liver steatosis, but major changes in the degree of lipid unsaturation were observed. On the other hand, for HF model more advanced steatosis was noticed with some modification in the degree of lipid unsaturation, in reference to the control.
9:55 Glucose Sensing as a Key Component in Future Treatment of Diabetes

Oliver Plettenburg

Head of Biosensors & Chemical Probes, Diabetes Research & Translational Medicine
  Diabetes is a pandemic chronic disease with more than 390 million people being affected worldwide. Today several drug classes are available to manage the disease, however only symptomatic treatment available.

In this presentation I will discuss the specific importance of thorough glucose control for treatment of Diabetes. I will highlight newly arising, device mediated options that are currently under development and bear the promise to significantly improve therapeutic outcome. This will in particular comprise new approaches for continuous blood glucose monitoring, as well as the utilization of glucose-sensing materials for smart delivery of insulin.
10:20 Morning Networking & Coffee Break
Optical and Single Molecule Sensing
Moderator: Craig Aspinwall, University of Arizona
11:00 Fluorescent Metal Nanoclusters-Based Sensing Systems for the Detection of Small Ions
Huan-Tsung Chang
Professor, Chemistry
National Taiwan University
Gold, silver, and copper nanoclusters (NCs) possessing photoluminescence have become popular sensing materials. Having advantages of long life time, large Stokes shift, stability, ease in preparation/conjugation, and biocompatibility, these NCs have been employed to develop sensitive and selective sensing systems for a variety of analytes, including proteins, DNA, organic molecules, and metal ions. In addition, they have been used in cell imaging. In my talk, I will present strategies for their preparation and their optical properties. To highlight practicality of these NCs, several interesting sensing systems for the detection of analytes such as H2S, CN-, and Hg2+ will be presented.
11:25 Enzymatic Tricks for Singe Molecule Genomics

Yuval Ebenstein

Principal Investigator
Tel Aviv University
Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material and is lacking in its ability to resolve variations between genomes. Chromosomes contain a plethora of variable regions that include single point mutations (SNP), structural variations (SV), copy number variations (CNV) and DNA repeats. In addition, the information content of the genome extends beyond the base sequence in the form of chemical modifications such as DNA methylation or DNA damage lesions. We show how DNA processing enzymes may be utilized to fluorescently label various genetic and epigenetic marks on DNA. By regarding chromosomes as single molecules and applying experimental principles of single molecule detection we gain access to the structural variation and long range patterns of genetic and epigenetic information. We demonstrate how physical extension of long DNA molecules on surfaces and in nanofluidic channels reveals such information in the form of a linear, optical “barcode”, like beads threaded on a string, where each bead represents a distinct type of observable. Recent results from our lab demonstrate our ability to detect the epigenetic mark 5-hydroxymethylcytosine and various forms of DNA damage on individual genomic DNA molecules. As well as detecting and quantifying DNA damage on the single molecule level.
11:50 Novel Strategies in Single Molecule Sensing
Joshua Edel
Reader, (Bio)Sensing & Analytical Sciences
Imperial College London
Analytical sensors play a crucial role in today’s highly demanding exploration and development of new detection strategies. Whether it be medicine, biochemistry, bioengineering, or analytical chemistry the goals are essentially the same: 1) improve sensitivity, 2) maximize throughput, 3) and reduce the instrumental footprint. In order to address these key challenges, the analytical community has borrowed technologies and design philosophies which have been used by the semiconductor industry over the past 20 years. By doing so, key technological advances have been made which include the miniaturization of sensors and signal processing components which allows for the efficient detection of nanoscale object. One can imagine that by decreasing the dimensions of a sensor to a scale similar to that of a nanoscale object, the ultimate in sensitivity can potentially be achieved - the detection of single molecules. This talk highlights novel strategies for the detection of single molecules using multiphase microfluidics.
12:15 Lunch on Your Own
Optical and Single Molecule Sensing (cont.)
Moderator: Nuno Reis, Loughborough University
2:10 DNA Origami Nanopores: Developments and Challenges
Ulrich Keyser
Reader, Experimental Physics, Cavendish Laboratory
University of Cambridge
DNA nanotechnology has enabled the construction of DNA origami nanopores. These synthetic designer nanopores promise improved capabilities for improved single molecule detection. Here, we will review the recent developments of DNA origami nanopores both in lipid and solid-state membranes. These structures have extraordinary versatility and are a new and powerful tool in nanobiotechnology for a wide range of important applications beyond molecular sensing. We discuss the current challenges and possible solutions that would enhance the sensing capabilities of DNA origami nanopores. Finally, we anticipate novel avenues for future research and highlight a range of exciting ideas and applications that could be explored in the near future.

Key benefits:
· Designer nanopores for single-molecule detection
· Design of man-made ion channels from DNA
· Novel tools for nanobiotechnology
· Future applications in nanomedicine to replace ion channels
· Simple fabrication of tailor-made ion channels from DNA
2:35 Detecting Specific Protein Binding and RNA Unfolding Pathways on Single RNA Molecules
Mark Williams
Professor, Physics
Northeastern University
Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC destabilizes nucleic acids, including the transactivation response (TAR) hairpin, to facilitate their structural rearrangement into the lowest free energy conformations. We combine single molecule optical tweezers measurements with a quantitative mfold-based model to characterize the equilibrium TAR stability and unfolding transition state for TAR RNA. Experiments show that adding NC preferentially destabilizes the upper part of the TAR hairpin and shifts the transition state closer to the bottom of the TAR stem. Incorporating TAR destabilization by NC into the model reveals that a subset of preferential protein binding sites is responsible for altering the unfolding landscape, and we quantify the destabilization induced at these specific sites. These results provide the first direct quantitative measurement of alterations in an RNA unfolding landscape induced by specific protein-RNA interactions and allow us to identify the specific binding events responsible for altering the unfolding landscape.

Benefits of talk:
· RNA destabilization and unfolding is critical for retroviral replication
· Single molecule RNA unfolding allows quantitative characterization of the unfolding landscape
· Detection of specific HIV nucleocapsid protein binding events is obtained by combining mfold theory with single molecule measurements
· Results for HIV-1 nucleocapsid protein may be useful for targeting this protein in anti-retroviral therapy
· Method can be generally used to characterize RNA-protein interactions critical for wide variety of biological processes
3:00 Identifying Bacterial Plasmids Coding for Antibiotic Resistance Using Optical DNA Mapping

Fredrik Westerlund
Associate Professor, Biology and Biotechnology
Chalmers University of Technology
The use, and overuse, of antibiotics over the last decade has led to a dramatic increase in antibiotic resistant bacteria. The World Health Organisation (WHO) has therefore warned that we are rapidly approaching a “post-antibiotic era” where infections that have been treatable for decades will become lethal. A large fraction of the genes coding for antibiotic resistance are located on bacterial plasmids, circular DNA molecules that are separated from the chromosomal DNA of the bacteria. Here, we present how optical mapping of single plasmids in nanofluidic channels as a novel approach for characterization of bacterial plasmids. Due to the miniscule amounts of sample needed for nanofluidics experiments it is possible to omit the time-consuming cultivation and amplification steps that are normally required for traditional plasmid identification.

The optical mapping assay is based on our previous work on competitive binding of two molecules to DNA, resulting in an intensity distribution along the DNA, a barcode, which reflects the underlying basepair sequence with kilobasepair (kbp) resolution. This assay allows us to identify bacterial plasmids from a database of all sequenced plasmids. We also demonstrate how we can use the barcodes to investigate the polyclonal spread of resistant bacteria during a resistance outbreak at a neonatal ward in Gothenburg. The single molecule nature of the assay means that it is perfectly suited when a fast answer is crucial, such as during sepsis or for patients with malfunctioning immune response.
3:25 Mid-Infrared Substrate-Integrated Hollow Waveguides: A New Generation of Breath Diagnostics

Boris Mizaikoff

Professor and Chair
Institute of Analytical and Bioanalytical Chemistry, University of Ulm
Mid-infrared (MIR; 3-20 µm) sensor technology is increasingly applied in biodiagnostics taking advantage of the inherent molecular specificity, which enables the discrimination of molecular constituents at ppm-ppb concentration levels.1 Substrate-integrated hollow waveguides (iHWGs) are layered structures providing light guiding channels integrated into a solid-state substrate material, which simultaneously serve as highly miniaturized and optically efficient gas cell.2 The unprecedented modularity of this novel waveguide approach facilitates tailoring iHWGs to almost any kind of gas sensor technology providing adaptability to the specific demands of a wide range of sensing scenarios and spectral domains. The analytical utility of such devices for advanced MIR gas sensing applications is discussed for the gaseous constituents butane, carbon dioxide, cyclopropane, isobutylene, and methane, as well as for IR sensors taking advantage of iHWGs in breath diagnostics (detecting 12CO2/13CO2 and isoprene),3-5 in environmental monitoring (detecting ozone),6 and for hazardous gas sensing (detecting H2S).7

Key benefits to the audience:
· The latest optical technology in breath diagnostics is presented
· Application of mid-infrared gas sensing for relevant breath constituents
· Ppm-ppb level detection limits in complex matrices (with/without preconcentration)
· Application of quantum cascade lasers (QCLs) with commercial potential
· Portable devices using broadband (FTIR) or broadly tunable laser (QCL) light sources
· Potential for on-chip miniaturization
3:50 Afternoon Networking & Coffee Break
Nanomaterial Enabled Sensors
Moderator: Hua-Zhong (Hogan) Yu, Simon Fraser University
4:20 Core-Shell Nanoscintillators: Towards Intracellular Detection of Radiolabeled Metabolites
Craig Aspinwall
Associate Professor, Chemistry and Biochemistry
University of Arizona
Temporally resolved, intracellular detection of carbohydrate messengers is limited by a dearth of optical indicators compatible with detection of these analytically challenging species. Traditional carbohydrate detection in biological systems has relied upon the use of radioisotope-labeled precursors, though detection of the resulting messengers is generally not possible in single living cells. To address this limitation, we have developed scintillating polystyrene-core silica-shell nanoparticles (nPs) for scintillation proximity assay (SPA) applications with b-particle emitting radionuclides in physiological samples and biological systems, with an eventual goal of single cell analysis. Development of a SPA particle platform requires the selection of component materials that offer high quantum yields, good sample compatibility (e.g., solubility), resistance to degradation from exposure to radiation, and highly specific and sensitive analyte binding. nanoSPA particles were prepared using ca. 150 nm scintillant-doped polystyrene core particles followed by addition of 15-25 nm thick silica shells. The resulting particles are non-toxic, easily functionalized with specific binding motifs that facilitate highly specific detection of target metabolites, even in the presence of background radioisotope, easily dispersed and serve to detect 3H, the most ubiquitous and low energy biologically-relevant isotope. Additionally, the high surface area to volume ratio provides broader dynamic ranges and higher efficiency than current SPA materials.

Benefits of talk:
· A new paradigm for intracellular analysis of challenging second messengers
· An approach that may complement metabolomics platforms
· A discussion of nanomaterial platforms for non-traditional sensing
· Long term clinical implications
4:45 Efficient Nanophotonic Biosensors for Low Cost Real-Time Lab-on-Chip Assays
Daniel Hill
Biosensors Leader, Group of Optical Spectroscopy of Solids and Soft Matter
Institut de Ciència dels Materials
Universitat de Valenci
In this presentation we overview advances in the development of efficient nanophotonic biosensors for low cost real-time lab-on-chip assays within four EC funded projects.

For ring resonator-based sensors, volumetric limits of detection (LoD) of 5x10-6 RIU (refractive index units) and 8.3x10
-6 RIU for sensitivities of 246nm/RIU and 2169nm/RIU were reported from FP6 SABIO (at 1.31µm) and FP7 InTopSens (at 1.55µm) respectively. These compare well to the state of art of 7.6×10-7 RIU for a sensitivity of 163 nm/RIU, as does the porous alumina- based membrane sensors in FP7 Positive with their LoD of 5x10-6 RIU. More interestingly for the membrane sensors the standard deviation of their measured values was below 5% and their flow through design with lateral distances to the sensor surface less than a diffusion length permit fast response times, short assay times and the use of small sample volumes (< 100 µl). For protein binding recognition, within SABIO a surface LoD of 0.9 pg/mm2 for anti-BSA on a gluteraldehyde-covered surface was recorded, corresponding to a 125ng/ml anti-BSA solution, whilst in InTopSens 5pg/mm2 and 10ng/ml for biotin on a streptavidin coated surface was seen. For an assay of b-lactoglobulin - anti-b-lactoglobulin - anti-rabbit-IgG –streptavidin conjugated CdSe quantum dots the positive sensors demonstrated a noise floor for individual measurements of 3.7ng/ml (25pM) for total assay times of under one hour.

· Demonstration of the advances in integrated silicon photonic biosensors
· A comparison of limits of detection for different biosensor technologies
· A focus on the importance of flow through sensor designs for fast response and short assays times, and the use of small volumes of expensive reagents.
· Potential for multi-parameter biosensing.
 5:10 [Short Oral Presentation from Exemplary Submitted Abstracts]
  Nanodiamond-Delivered, Antisense RNA Inhibits the Progression of Colon Cancer Cells
Veronika Benson
Head of the Laboratory
Laboratory of Molecular Biology and Immunology, Institute of Microbiology of the ASCR
For the successful development of a therapeutic nanosensor, limiting factors are primarily the uptake of a functionalized nanoparticle by live cells and the sufficient release of effector molecules. Short microRNA molecules regulate most cellular processes, and their dysregulation leads to a disease burden; thus making them important therapeutic targets. The aims of our study were to a) evaluate the efficiency of RNA interference, triggered by short antisense RNA linked to a fluorescent nanodiamond; b) define the means of transport via the cell membrane; c) confirm the release of effector RNA in the cytoplasm; and d) detect the inhibition of target oncogenic microRNA-21 in colon cancer cells. To meet our goals we coated fluorescent nanodiamonds (FND) 50nm in size with poly-ethylene-imine and 22bp long RNA complementary to microRNA-21 (miR-21). A FND-carrier enabled us to monitor the successful delivery and release of the carried antisense RNA into the cytoplasm of colon cancer cells, as confirmed by confocal microscopy. Regarding the means of transport, both endocytosis and micropinocytosis were involved in the internalization of the functionalized complexes. The introduction of the antisense RNA triggered strong RNA interference, as revealed by the significant abolition of target microRNA-21. As a consequence of the elimination of microRNA-21, we observed changes of the mRNA level of PDCD4 and Bax, which are key miR-21 target genes, important in the regulation of apoptotic cell death. Changes in miR-21 and mRNAs expression were detected by real-time RT-PCR, with a specific TaqMan probe. Finally, we confirmed the therapeutic efficiency of our FND-antisense RNA complex in colon cancer cells by observing the induction of cell death mediated by caspases 3 and 7, the changes in cytoskeleton reorganization, and the inhibition of cancer cell migration. In summary, we have proven that fluorescent nanodiamonds, functionalized with a polymer and an antisense RNA trigger specific RNA interference, subsequently reduce cancer cell mobility and drive these cells to an apoptotic cell death. Such effect makes them promising therapeutic nanosensors. This work has been funded by the ESF and the Ministry of Education of the Czech Republic: Nanointegrace CZ.1.07/2.3.00/20.0306.
5:25 Evening Networking Reception & Poster Session

Day 1 Day 2
Day 2 - Wednesday, May 6, 2015
7:30 Continental Breakfast
8:00 Welcoming Remarks
Point-of-Care Sensors
Moderator: Alexis Vallée-Bélisle, Université de Montréal
8:05 Computer-Readable BioDiscs for Molecular Diagnostics
Hua-Zhong (Hogan) Yu
Professor, Chemistry
Simon Fraser University
Current guidelines for healthcare emphasize rapid testing and reporting, which can be better satisfied by near-patient or at-home point-of-care (POC) diagnosis rather than centralized medical laboratories using automated multi-analyte analyzers by trained professionals. With POC testing, the turn-around time will be significantly reduced, which ultimately leads to earlier decision making and more efficient medical treatment. At present, POC protocols are predominantly based on rapid-test immunoassay strips/cassettes, i.e., lateral-flow immunochromatographic assays that combine gold nanoparticles for colorimetric (mostly qualitative) detection and flow-through systems for sample delivery.

Compact disc (CD) technology is a promising alternative to today’s POC testing protocol, particularly in the development of a new generation diagnostic tools for on-site and at-home use. We have established both the surface chemistry and signal readout method for fabricating and running disc (CD-R, DVD)-based bioassays for various molecular analytes; these bioDiscs can be read quantitatively with standard computer drives and stand-alone disc players. Because no modification to either the hardware or the software driver is needed, this research promises a platform POC technology for rapid, low-cost, and high-throughput medical diagnosis.

(1) Yu, H.-Z.; Li, Y.; Ou, L.M.L. Acc. Chem. Res. 2013, 46, 258-268.
(2) Li, Y.; Ou, L. M.-L.; Yu, H.-Z. Anal. Chem. 2008, 80, 8216-8223.
(3) Wang, H.; Ou, L. M.-L.; Suo, Y.; Yu, H.-Z. Anal. Chem. 2011, 83, 1557-1563.
(4) Li, X.; Shi, M.; Cui, C.; Yu, H.-Z. Anal. Chem. 2014, 86, 8922-8926.
8:30 A “Lab-on-a-Stick” Approach to Point-of-Care Testing

Nuno Reis
Lecturer, Chemical Engineering
Loughborough University

There is a major need in the multi-billion dollar point-of-care (POC) testing market for multiplex tests that combine the simplicity and portability of immunochromatographic lateral-flow assays with the sensitivity and quantitative capability of sophisticated clinical laboratory equipment. This presentation will give an overview into the development and application of a new generation of simple, affordable and scalable microfluidic devices to sensitive and quantitative assays. We will demonstrate that the surface properties and refractive index of microeengineered fluoropolymer material allows combining conventional assay chemistry with the unique signal-to-noise ratio in the microfluidic material for rapid optical colourimetric or fluorescence detection using naked-eye or very low-cost optoelectronic components, including smartphone technology. We have established the principles for sensitive (femtomolar) immunoassay detection of cytokines and cancer and cardiac biomarkers from whole blood, and will show for the first time the application of our new “Lab-on-a-stick” concept to instantaneous blood typing and other multiplex assays.

Key benefits to the audience:
· The latest achievements in developing a new scalable and cost-effective microfluidic platform combining optical smartphone detection and conventional assay chemistry
· Application of these novel fluoropolymer microfluidic to rapid and miniaturized immunoassays:
- Femtomolar quantitation of multiple cytokines
- Portable PSA detection from whole blood using smartphone technology
· Quantitation of a range of cardiac biomarkers in singleplex and multiplex formats
· A novel “lab-on-a-stick” concept will also be presented
8:55 Engineering Biomolecular Switches for Antibody Sensing and Actuation
Maarten Merkx
Associate Professor of Protein Engineering, Department of Biomedical Engineering and Institute of Complex Molecular Systems
Eindhoven University of Technology
Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Antibodies are important biomarkers in broad range of diseases, and particularly important for the diagnosis and surveillance of infectious diseases, autoimmune diseases and allergies. In addition, antibody-based drug therapies constitute an important part of newly introduced drugs, most importantly in the field of oncology and inflammatory diseases. From a molecular engineering perspective, antibodies are attractive because there characteristic Y-shaped presentation of (at least) two antigen binding domains allows for the development of generic biomolecular switch mechanisms. In my lecture I’ll show how these unique structural features of antibodies can be harnessed to develop new concepts for point-of-care antibody diagnostics and the control of antibody-based targeting. Examples include the development of switchable reporter enzymes that allow simple colorimetric or luminescent detection of antibodies directly in solution and the use of bivalent peptide-DNA conjugates as easily applicable molecular locks to control antibody activity using DNA-based logic gates.

Biosensors that allow specific antibody directly in solution
Modular engineering principles allow exchange of reporter function and/or antibody-recognition domains in a plug-and-play type fashion
Bivalent peptide-DNA conjugates provide easily applicable molecular locks to reversibly control activity of monoclonal antibodies
Bivalent peptide-DNA locks provide a generic link between DNA-based molecular computing and antibody-based diagnostics
9:20 Nature-Inspired Switch-Based Sensors for the One-Step Detection Directly in Whole Blood
Alexis Vallée-Bélisle
Assistant Professor, Chemistry
Université de Montréal
All creatures, from bacteria to humans, monitor their environments in order to survive. They do so with biomolecular switches, made from RNA or proteins. Inspired by this natural technology, our laboratory has developed several new artificial biosensors that use fluorescent or electrochemical nanoswitches made from DNA scaffolds that measure temperature, pH, or various chemicals ranging from small molecules (e.g. drugs, explosives) to various macromolecules such as transcription factors and antibodies. These sensors are reagentless and selective enough to be employed directly in complex samples such as cellular extracts and whole blood. In my talk, I will explain how we design and build these nanoswitches and show how a better understanding of natural biomolecular switches and their mechanisms of regulation (e.g. inhibition, activation, and cooperativity) significantly helps our efforts to build more efficient sensing technologies.
9:45 DNA-Based Nanoswitches for Clinical Diagnostic Applications
Francesco Ricci
Associate Professor, Department of Chemistry
University of Rome, Tor Vergata
Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamp like embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions.

By taking advantage of the pH-dependence of parallel Hoogsteen interactions we have also rationally designed a general strategy to re-engineer nanoswitches whose opening and closing can be controlled by pH. More specifically, we have re-engineered DNA-based switches so that their sequence contains a triplex-forming portion that can be folded and unfolded over different pH windows. Such strategy can be of utility as a pH nanometer and for controlling release of drugs in a pH-dependent fashion.

1) Given their advantageous attributes in terms of affinity and specificity clamp-switches will be of utility for sensing applications
2) pH-nanoswitches can sense pH over more than 5 units of pH
3) These swiches can find applications not only in sensing but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.
10:10 Morning Networking & Coffee Break
Lab-on-a-Chip, Microfluidics, and Nanofluidics
Moderator: Fredrik Westerlund, Chalmers University of Technology
10:45 Nanomechanical Methods for the Analysis of Protein Aggregates
Thomas Burg
Research Group Leader
Max Planck Institute for Biophysical Chemistry
In this talk, I will describe new approaches of our laboratory towards the characterization and separation of nanoparticles and biomolecular complexes by nanomechanical methods. In particular, methods for studying the kinetics of amyloid formation will be described. Amyloids represent an important class of protein aggregates, which are associated with a wide range of human diseases and with the degradation of biotechnological products. Experimental investigations of the pathways of amyloid formation are often hindered by a lack of sensitive and quantitative methods that can be used with small volumes. In this talk, I will describe some routes to address this problem using a combination of micro-/nano-electromechanical systems and microfluidics. Embedded channel microresonators of only 10 pL volume are shown to enable label-free, mass-based measurements of protein aggregation kinetics. The sensitivity of the method is greatly enhanced by a fluctuation analysis termed mass correlation spectroscopy (MCS). This technique has been used to monitor the formation of insulin amyloids from monomers to mature fibrils. While, during MCS measurements, molecules are dispersed free in solution, embedded channel resonators also can be used in a surface-based mode analogous to the quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). In this mode, the devices are advantageous due to their low sample consumption, wide dynamic range, and reaction limited kinetic measurements. The benefits and limitations of nanomechanical mass measurements and other nanofluidic techniques for the analysis of large biomolecular complexes will be discussed.

· New label-free, highly sensitive methods for detection of protein aggregates are described
· The potential and limitations microfluidic techniques for analysis of biomolecular complexes is shown
· Discussion of surface modification issues in micro-/nanofluidic systems and capillary devices
· Applications of microfluidic mass sensors to measuring amyloid formation kinetics are presented
11:10 Early Disease Diagnosis Using Nanoplasmonics-Based Optical Sensor
Jaebum Choo
Professor, Bionano Technology
Hanyang University
We report the development of a programmable and fully automatic microfluidic sensor that integrates a gradient microfluidic device with gold-patterned plasmonic sensing platforms. This device provides a convenient and reproducible nanoplasmonics-based immunoassay platform for various biomarkers. The utility of this platform is demonstrated by the quantitative immunoassay of various cancer and cardiovascular protein markers. Our proposed nanoplasmonics-based immunoassay platform has many advantages over other immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with mixing stages. The total assay time from serial dilution to nanoplasmonics detection takes less than 30 minute because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for early disease diagnosis.

Benefits of talk:
• Development of a novel blood test technique using nanoplasmonics-based point-of-care sensor
Application of nanoplasmonics sensor for highly sensitive biomedical analysis
Bioconjugation of plasmonic nano tags for specific biomarker targeting
Innovative nanoplasmonics-Based microfluidic devices and lateral flow assay kit
Clinical applications of nanoplasmonics-based optical sensor
11:35 Microfluidic Platform for Experimentation on Bilayer Lipid Membranes and Pore-forming Species

Séverine Le Gac
Associate Professor, MESA+ Program director Nanomedicine
MESA+ Institute for Nanotechnology, University of Twente
Membrane proteins are key-targets for the development of new drugs, and dedicated platforms are desired for high throughput screening of drugs, and where those proteins are in a simplified yet functional environment. The combination of microfluidics with bilayer lipid membrane (BLM) models is particularly appealing to reach this goal: miniaturization brings enhanced electrical capabilities and higher BLM stability, and microfluidics is ideal for automation and multiplexing.

We have developed a microfluidic platform for experimentation on BLMs and ion channel proteins (Stimberg et al., Small 2013), which consists of two glass substrates separated by a Teflon film. The glass substrates house two independent microchannels, representing the intra- and extra-cellular environments, as well as fluidic accesses, while the Teflon film includes a micrometer-sized aperture across which a BLM is formed. BLMs are formed by successive flushing of a lipid solution in n-decane and measurement buffer. BLMs exhibit excellent and reproducible seal resistance (> 10 G?), membrane capacitance (20-30 pF for 100-?m apertures), and specific capacitance in the range of 0.50-0.75 ?F/cm2, depending on the BLM composition.

We will present different sets of experiments conducted in our microfluidic platform including electrophysiological measurements on model pore-forming species (gramicidin and ?-hemolysin) and KcsA potassium channels, as well as studies on the relationship between membrane properties (fluidity and thickness) and ion channel activity using a unique combination of confocal imaging and electrophysiological measurements. Lastly, we will discuss ongoing technological developments on multiplexing of the device and automation of the experimentation.
12:00 Portable Droplet Microdialysis for Continuous Chemical Monitoring
Xize Niu
Associate Professor, Electromechanical Engineering
University of Southampton
Continuous measurement of biomolecule/drug concentrations directly from tissue fluids offers the exciting possibility of understanding physiological or pathological processes, recording responses to stimuli, drug metabolism, and even developing new therapies that use biomarker levels to guide treatment in real time. However, such measurement is challenging - the fluids are complex mixtures, the volumes can be very small, and detection methods are limited. Here we have developed an enabling portable sensor device to tackle this challenge. The device combines microdialysis and droplet microfluidic techniques, can sample body fluids into nano litre droplets, perform assays and measurements in situ, and communicate wirelessly to the user. We envisage this novel technology will revolutionize the current practices of sampling and chemical sensing, and find broad applications in disease diagnostics and monitoring, drug development, organ transplantation and the other areas. I will also discuss the challenges and potential solutions on analyzing minute amount of complex biological fluids.

Benefits of talk:
· How can microfluidics address the challenges of continuous monitoring of body chemicals?
· Invasive/semi-invasive/non-invasive?
· The essential elements and special considerations for a portable/wearable sensor device?
12:25 Lunch Provided by GTCbio

Conference Concludes

Day 1 Day 2