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About the FluoroMag E.U. Consortium

Consortium Members:

• Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
• University of Twente, Enschede, The Netherlands
• University of Santiago de Compostela, Santiago de Compostela, Spain
• Nottingham Trent University, UK [[http://www.ntu.ac.uk/apps/Profiles/50684-1-9/Dr_Quentin_Hanley.aspx
• Nanogap, Santiago de Compostela, Spain

Multiparameter sensing for high sensitivity diagnostics using fluorescent and magnetic nanoparticles

As part of the EU FP6 research framework in the field of genomics and biotechnology for health, a new consortium “FLUOROMAG” coordinated by Dr. Donna Arndt-Jovin, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, will develop new diagnostic tools for use in tumor biology and the detection of very low levels of pandemic viruses.

Contents

Introduction

General Overview

Attach:
The project of the consortium has two elements. The first was the development of new, small NPs, i.e. with sizes below 10 nm (less than a millionth of a cm): “nanodots” and magnetic NPs. These new nanodots were tested for their characteristics and behavior for applications in Chemistry and Biomedicine. The consortium also developed targeted NPs and quantum dots will be derivatized for specific recognition of biomolecules such as tumor markers (for breast cancer and brain tumors). Other core-shell “onion-like” NPs developed by the partner in Santiago de Compostela have diverse and strong magnetic properties and will be tested for their application in micro-chip and MRI diagnostics.

In a parallel effort, several of the partners optimized the design and performance of a new type of high-speed, sensitive, optically sectioning microscope known as the Programmable Array Microscope (PAM), for use in both the basic research and medical communities. The PAM is very versatile in that it implements many imaging modalities and has been under development in the Molecular Biology Dept. for the past 10 years. It has single-NP sensitivity, and is ideally suited for measurements of thick samples such as tissue slices and patterned arrays, important objects for diagnostic tests.

The FLUOROMAG consortium was awarded € 2.5 million by the European Union for a period of 3 years. The research project leaders of the consortium are: Donna Arndt-Jovin (MPIbpc, Germany), Arturo López-Quintela (Univ. of Santiago de Compostela, Spain); Vinod Subramaniam (Univ. of Twente, The Netherlands); Quentin Hanley (Univ. of Nottingham Trent, UK). An SME is included in the consortium, Nanogap Sub-nm-powder SA, Spain (Tatiana López del Rio), who can produce the NPs in large scale.

Multiparameter sensing for high sensitivity diagnostics using fluorescent and magnetic nanoparticles
Consortium Members:

• Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
• University of Twente, Enschede, The Netherlands
• University of Santiago de Compostela, Santiago de Compostela, Spain
• Nottingham Trent University, Nottingham, United Kingdom
• NANOGAP subnm Powders , SA, Santiago de Compostela, Spain

Specific objectives:

(a) to produce nanoclusters or nanodots (NDs), and core-shell (CSs) nanoparticles of uniform size distributions and transfer of this technology to Nanogap, SA to scale up the synthesis of these nanoparticles for commercial production as well as supply the consortium with NPs for characterization of their extinction, fluorescent and magnetic properties and the further development of diagnostic tests.

(b) to devise conjugation strategies to couple biomolecules to NDs and commercially available quantum dots, (QDs) to produce probes that can specifically target macromolecules such as proteins and DNA/RNA in vitro and in cells and tissues.

(c) to develop multiparametric diagnostic assays using combinations of bioconjugated QDs and NDs as novel, fluorescent probes, and bioconjugated noble metal nanoparticles as extinction probes. The goal is to achieve high sensitivity and molecular and cellular recognition; to develop new diagnostic tests for tumors expressing high levels of Her2 and/or Her1.

(d) to develop a low-cost programmable array microscope (PAM) module for wide field microscopes which utilizes a spatial light modulator for illumination and emission to achieve high-speed sectioning and simultaneous measurement of multiple fluorescence modalities as a detection system for single and multiplexed diagnostic assays using nanoparticles developed in a-c. The research consortium will test and improve the capabilities of beta versions of this instrument for both research and clinical laboratories, achieving a demonstrator by the end of the project that could be commercialized.

Summary

The objectives of the consortium have met all of the targets outlined above.
Patent applications have been granted and/or applied for in the following areas:
(i) design and construction of a demonstrator Progammable Array Microscope (PAM) with new capabilities; (ii) the production of atomic clusters of nanoparticles for medical and industrial uses; (iii) a new diagnostic assay

Experimental results from research supported by the grant have been and continue to be published in peer-reviewed journals (30 to date) and over 300 talks or posters were presented at a wide variety of national and international meetings.

Nanoparticle probes

Objectives:

In work packages 1 to 3 the consortium agreed to produce, by unique emulsion and electrochemical procedures, well-defined noble metal nanodots (NDs) and core-shell nanoparticles (CSs), as well as conjugation strategies to link both commercial and newly developed nanoparticles to biomolecules to produce probes that could specifically target macromolecules such as proteins and DNA/RNA in vitro and in cells and tissues. The particle synthesis strategies were scaled to commercial production by Nanogap, P6. Prototype particles were made available to other members of the consortium throughout the contract.

Results:

As detailed in the WP1, a large number of different NDs have been produced and characterized by USC, Partner 3 with assistance from P6 (Nanogap for synthesis of particles for the partners). 45 nm gold particles were synthesized for P2 and P4 and characterized by P3 (see WP8 as well). Gold coated iron NPs were produced and characterized with the assistance of P1 (MPIBPC). 16 nm Fe@Au particles were produced for P2 (Twente) and bioconjugated in their laboratory (see WP8). Several methods of synthesis were explored for making magnetic gold, magnetic silica and cold-coated magnetic silica NPs. P6 was able to optimize the production of 8 nm magnetite and subsequently coat it with smaller gold NPs.

The most successful particles produced in the project are metallic clusters of Au, Ag, nd Cu, of few atoms. Cu clusters of 2-25 atoms were photoluminescent with stable emission for more than 2 years. Over a hundred different experimental conditions were explored for Au atomic clusters as well as different capping compounds. Size could be controlled by the conditions of the electrochemistry synthesis. The effect of temperature on the cluster size as well as the stability of the particle size was investigated, producing a variety of different photoluminescent particles. Ag clusters were obtained that had 15-50 atoms and emissions as high as 450 nm.

The synthesis methods did not produce as monodisperse particles as desired. Different procedures for purification of the Ag and Au atomic clusters to homogeneous preparations were investigated and ultimately HPLC was found to produce the highest yields and the best uniformity of cluster size. In addition it was discovered that the small atomic clusters had antibiotic character. This information was expanded into toxicity studies after successful preparation of larger batches of these particles and IP protection was applied for both on the synthesis routes as well as on applications of the atomic quantum clusters (AQCs). An additional interesting spin-off from this work has been the formulation of “electrical conducting ink formulations” based on mixtures of AQCs and nanoparticles. These materials are of particular interest to the electronic industry and their production has been transferred to the SME, P6 after obtaining IP protection. Finally, P1 is exploring the use of some of the AQCs as catalysts for electronic, optical and material industries.
Synthesis methods of silica and noble metal particles with high fluorescence quantum yields for bioconjugation in diagnostics proved elusive. Instead the synthesis and production of core-shell magnetic nanoparticles with a number of different techniques were investigated yielding a range of interesting products. In particular, gold-coated Fe3O4 were synthesized and characterized and supplied to P2 (Twente) for bioconjugation and characterization in a variety of different systems.

Partner 1, MPIBPC, directed WP2 and produced a procedure for the production of truly monovalent bioconjugation to quantum dots. There have been no synthesis routes published for such particles and this work will be soon in press. P1 successfully developed 4 other kinds of nanoparticles, bioconjugated to cell targeting molecules such as growth factors, antibodies, and affibodies and developed specific diagnostic tests with two of these during this project. All of these particles could be shown to target specifically cells and tissues with the expressed targets and to be effective in distinguishing cancer cells from normal cells. Some of the nanoparticles were also loaded with tumor inhibitory prodrug constructs, RNA and /or DNA as well as growth inhibitors. (a) Targeted magnetic switch particles were produced by synthesizing SPIONs (superparamagnetic iron oxide nanoparticles) doped with fluorescent molecules and bioconjugated to cell-targeting molecules. Activation and internalization could be induced by application of a magnetic field. These particles have the potential to be able to home to tumors and then to be induced to internalize by a directed magnetic field. The data on these particles will soon be submitted for publication. (b) Dual-fluorescent nanoparticle liposome carriers for DNA and RNA (or drug) delivery were formulated. They were shown to be excellent new tools for determining quantitatively the uptake and release of cargo, an interesting new diagnostic tool. The formulation and results of tests on tumor cells has been recently published in Bioconjugate Chemistry. (c) Bioconjugated and specifically targeted quantum dots were successfully developed for WP6 for diagnostic tests for tumors expressing Her1 and Her2 and the final formulations are the subject of an application for Phase 1 clinical testing and a major publication publsished in PLoS ONE showing for the first time a diagnostic tool for low-grade glioma tumors that show no gadolinium positive signal by MRI. (see WP6). (d) Microcapsule carrier particles were added to the program in the last 12 months of the project after an extensive study of uptake of affibody-enzyme conjugates by breast cancer cells expressing Her2. These microcapsules have the capacity to deliver up to a million enzyme molecules engineered to efficiently convert prodrugs to nucleotide analogs that inhibit cell proliferation. Preliminary studies show very promising results for specific targeting with these new constructs.

As described in WP3, partner 6, the SME NANOGAP, produced an extensive market analysis for NDs and CSs and their applications as well as an assessment of the molecular diagnostic market. In particular important contacts were established through fairs and expositions for products from the industrial scale production of particles developed in WP1 and spun off to P6. P6 also supplied the consortium with CS and ND particles for testing throughout the project. They have also led the way on the synthesis of Si NDs as possible alternatives to the noble metal NDs. A palette of commercial products has been scaled up for industrial production from the original formulations developed in WP1. These include silver nanoparticles (1) NGAP AQC Ag-1101-W as an anti-microbial additive; (2) NGAP AQC Ag-1102-W for biological applications since the atomic clusters have photoluminescence in the UV range; and (3) NGAP AQC Ag-1103-A, an acrylate dispersion of the atomic clusters that finds industrial use in the fabrication of medical equipment. The fluorescent properties of these formulations suggest that they might be useful in diagnostics if they can be transferred to an aqueous environment and successfully protected from oxidation. P6 has also produced iron oxide particles of about 10 nm as water soluble dispersions NGAP NP FeO-2202-AB that can be used in subsequent bioconjugation schemes (see WP2). Subsequent coating of the FeO NP by a silica shell results in 20 or 40 nm particles (NGAP NP FeO-2206-W and NGAP NP FeO-2207-W) that have also been coated with gold clusters by P3.
All of the deliverables anticipated in all 3 WPs were successfully completed.

Nanoparticle diagnostics

Objectives:

In work packages 4 through 9 the consortium should develop improved multiparameter diagnostic assays using bioconjugated commercial quantum dots (QDs), as well as consortium-produced NDs and absorbing NPs for viremias, for human erbB (Her)-expressing tumors and for general antibody capture assays. The possibility to extend the sensitivity of these diagnostic assays to the single molecule or single particle level was to be explored. Benchmark studies were to be carried out in the first phase of the project and preclinical protocols developed by the end of the contract.

Results:

Partner 4, NTU made benchmark assessments of a number of assays in the first phase of the project. In the second phase, HCV was assessed for sensitivity using a confocal assay (WP4). The confocal assay data resulted in the application of a patent for the procedure by P4. Extension of the assay to PAM measurements was precluded in Phase 1 by late delivery of the beta PAM module and difficulties with its performance. The assays were adapted to the PAM in phase 2.. These results have been published in the Journal of Biomedical Optics. Studies of the kinetics of antibody binding including comparisons of QDs and conventional fluorophores were carried out. A unique Z-axis multiplexing assay was devised and evaluated showing that up to 5 layers of 30 µm layers of glass or mica immunoassays could be resolved by confocal imaging systems be they raster scanning or PAM systems. All the deliverable of this WP were achieved.

In WP5 an improved assay for HCV was the goal by the end of the project. Because of constraints in the facilities available to the consortium, actual infective viruses could not be handled. After successful nanoparticle-PNA-branched DNA probe development in phase 1 the project focused on antigen recognition in phase 2. It was possible in the 2nd period to achieve a confocal sandwich assay for the detection of HCV in both a direct assay as well as an indirect assay. Single viral particle sensitivity was shown by the PAM sensitivity for detecting and tracking single quantum dots that were specifically targeted to cell surface receptors at high speed (image acquisition in 16 ms). Some of these data were published in a book Single molecule dynamics in life sciences (Wiley, 2009). Single particle tracking data have been submitted for publication.

WP6 lead by partner 1 (MPIBPC) to create improved diagnostics for human erbB over-expressing tumors was highly successful. P1 in collaboration with neurosurgeons at the University of Göttingen Medical School produced specifically conjugated quantum dots that targeted Her1 expression which is upregulated on greater than 60% of all human gliomal brain tumors. Both Mab- and EGF- (epidermal growth factor) coupled QDs specifically recognized not only high-grade glioma biopsies but also were able to distinguish tumor cells in low-grade biospies where no gadolinium positive signal can be visualized. Excellent delineation of tumor tissue from normal brain in fresh biopsies was achieved even in the case of low-grade tumors. The results of the high grade tumor protocol was published in 2009 in IEEE Transactions in Nanobioscience and the protocol for low-grade tumors was published in PLoS ONE in June 2010. The success of the protocol has allowed the clinical collaborators to propose a Phase 1 assessment of this diagnostic for patients in the near future. This proposal is being submitted to the advisory board of the Georg-August University Clinic in Gottingen, Germany for approval.
P1 was able to target both resistant and sensitive Her2 expressing breast tumor cells with specific affibody-conjugated probes formulated in the MPBPC. A conjugated prodrug converting enzyme and affibody construct were internalized by the Her2 expressing tumors. In order to increase the level of enzyme available for prodrug conversion a microcapsule technique was developed in the last 12 months of the project. The work on this nanoparticle is on-going and the goal is to produce a vehicle that would target the Her2 cells and make them more sensitive to chemotherapy.
All deliverables were achieved ahead of schedule for WP6 and the clinical application of the diagnostics developed in the project will benefit the citizens of the EU by helping to raise life-expectancy for patients with glioblastoma brain tumors.

A. T-1weighted MRI brain scan with no gadolinium positive signal of a grade II oligodendroma. (B-E) Digital macrophotographic images of ex vivo stained biopsies from the resected tumor stained with targeted (B-D) QD probes or (E) untargeted QDs taken with the same magnification and the same exposure times.

Partner 4, (NTU) directed WP7 to develop and demonstrate the use of multiplexed nanoparticle (NP) based assays using PAM detection by the quantitative separation of fluorescent signals. P4, NTU, was able to benchmark combinations of dyes in Phase 1 and extended this to a quantitative, analytical equation capable of separating pH sensitive dyes and nanoparticles. P4 has successfully synthesized new phosphorescent coupled NPs that were useful in designing an assay for multiplexing different antibodies simultaneously. The work is currently in press in The Analyst. In addition a multiplexed nanoparticle assay for dengue fever was achieved using multiple endpoints.

As a collaborative part of the workpackage P4 and P1 worked together at the MPIBPC to record full spectral images on the PAM. The spectroscopic PAM was able to resolve mixtures of quantum dots and complex fluorescent biological samples. Both 405 and 488 nm excitations were used. New software was developed by P1 for displaying the data from the spectroscopic PAM. Thus all deliverables were met.

WP8 led by Partner 2 (Twente) developed an extremely sensitive protein -based sensor with gold NPs (diameter 25-50 nm) and gold-coated magnetic Core-shell particles (magCSs). Partner 2, Twente, devised assays for gold NPs by the following approaches: (a) colorimetric (dark field microscope with color camera); (b) spectroscopic (spectrograph+camera); and (c) bulk absorption (spectrophotometer). A flow-through system based on thin-layer flow cells was successfully developed by P2 that can detect binding of hapten to pre-functionalized particles. In phase 2 of the project the coupling of various proteins to the gold NPs was improved by linking the gold to the protein through a thiol alkyl chain with a terminal amino group on an oligoethylene glycol. Such a gold surface derivatization rendered the spectroscopic properties of the particles stable and created a versatile linkage chemistry. In darkfield binding assays, the flow system developed by P2 was able to measure single binding events. The deliverables were met.

WP9 was lead by Partner 2 (Twente) and focused on the effective use of magnetic nanoparticles (magNPs) for inter- and intracellular manipulations, sensing and separation. P2, Twente, derivatized magNPs for intracellular biosensor applications. The cellular phagocytotic pathway has been used as a model system for testing pH biosensor magNPs and manipulating them inside living cells with magnetic tweezers constructed in the laboratory of P2. In order to understand better the types of possible manipulations that could be achieved P2 calculated the magnetic force on a 50 nm Cobalt particle as function of distance to the magnetic pole for different types of pole geometries and derived parameters for long range/low force regime up to the short range/ high force regimes. In collaboration with P1, (MPIBPC) who synthesized superparamagnetic iron oxide nanoparticles (SPION) of ~10 nm mean size, and bioconjugated them with targeting antibodies to Her1 and Her2 (see WP2 and WP6), studies on the particles were undertaken to understand the force and duration of magnetic attraction. Systematic investigation of the effects of magnetic field strength, duration and direction on activation of Her1 was undertaken. Interesting new information on growth factor activation has resulted from these successful experiments that will be published in the near future from both groups. P6 and P3 supplied various core-shell nanoparticles to the project.

Development of high-speed, multiparameter detection of NPs with a consortium-optimized Programmable Array Microscope (PAM)

Objectives:

The goal of the consortium was to have developed a demonstrator for a commercial PAM instrument, optimized for the healthcare community, to make sensitive diagnostic measurements by the end of the project.

Results:

Partner 1 (MPIBPC) undertook the responsibility for WP10 at month 16 due to loss of partner 5 to achieve the original goals set out in the contract with the EU to design and bulid a demonstrator PAM that shows the capabilities and superior performance of such a microscope for high-speed optical sectioning. A successful redesign of the hardware and optics for a new Generation 3 PAM with superior capability was achieved

The new Generation 3 PAM design consists of the following elements:

1. Use of a TI DMD digital micromirror device as the SLM control element with Visitech control electronics and software
2. Telecentric relay between microscope and DMD.
3. DMD condensor from DMD to secondary afocal relay.
4. Afocal telecentric relay from DMD to CCD.
5. Image combiner and CCD relay.

Since the PAM microscope utilizes the SLM in both the excitation and emission pathways the optical path presents unique problems for maintaining high resolution as well as achromatic aberration-free imaging. In addition, since the DMD functions by deflecting the “on” and “off” pixels at an angle of +/- 12º, the imaging beam will have an angle of 24º to the optical axis. A unique solution to this optical problem was achieved by the scientists at P1 and this optical design has been protected by a patent applications, EP 1000 3066.7, USP 61/316,671. Careful selection of glass for the optical components has assured that the final demonstrator has the best properties for imaging with single molecule sensitivity. The completion of a demonstrator unit was achieved at month 42, thus achieving the goal of the project. A European SME has signed a ‘Letter of Intent’ and is building a second demonstrator with the commitment to start commercial production of the PAM3 in late 2010/early 2011.

PAM v3 demonstrator - covers and baffles removed to show optical components more clearly; optical paths drawn in green.

New software capabilities have been generated for the PAM. In phase 1 of the project the real-time imaging control software was developed by P1 as well as software to interleave multiple light sources and to take multiple emission wavelength images. Pattern interleaving was developed as well. In Phase 2 P1 created a new n-dimensional image rendering software package to visualize the often four- or five-dimensional data generated by the PAM. This software is able to show arbitrary projections of large (multi-GB) data sets interactively. In addition, it can perform spectral rendering for the images taken for WP7. Arbitrary combinations of linked XY/XZ/YZ or XY/XZ/XC projections are possible. Other capabilities include length measurements and histogram analysis.

The goal of WP11 was to investigate the incorporation of other detection modalities besides intensity and location into the PAM imaging capability. Partner 1, MPIBPC has led this effort. In phase 1 LED light sources were developed for bright samples. These were improved in phase 2 by using photonic light emitting surfaces called Phlatlights. P1 demonstrated single-molecule sensitivity with 16 ms acquisition using laser light sources for 1k x 1k pixel images and faster acquisition for regions of interest (ROIs). In phase 1 lifetime imaging with the PAM was realized with incorporation of a phase modulated light source and camera system in collaboration with Lambert Instruments.

In phase 2 of the project several more new capabilities were realized for PAM imaging. The goal of high-speed imaging in live cells is often to collect data sets over long time periods in order to determine the effects of drugs, inhibitors or stimulants on the cells. Such studies have usually resorted to non-confocal methods in order to reduce bleaching and detrimental effects of high excitation intensities. The PAM offers a unique capability for such measurements since the instrument itself can regulate the light dosage so as to avoid bleaching and toxicity. The MLE-PAM (minimized light exposure PAM) is described in detail in the report for WP11. In short, this software allows imaging of living embryos and cells over periods of hours instead of 10s of minutes. In addition, because the imaging is under computer control, cells or sub-cellular compartments that are moving over the time of the experiment can be tracked and kept in focus without operator intervention. These new capabilities of the PAM have been recently published in an article in the J. of Microscopy. WP6 has utilized PAM imaging for tumor biopsies and shown the improved capabilities for distinguishing localized tumor cells with 3-D imaging rather than 2-D imaging. These data were recently published in PLoS ONE and suggest that for a number of applications in cell biology the commercialization of the PAM could include a suite of specialized instruments as well as the highly versatile PAM for the basic research market. Instruments could be targeted for the health care market for specific diagnostic tests, such as an operating theater PAM, (see WP6).