INFLUENZA - Influenza virus integrated detection platform.
Background
A new diagnostic tool for influenza virus detection based on magnetic nanoparticle markers and highly
sensitive magnetic field sensors is proposed. Traditional diagnostic methods for human influenza virus
infections are based on cell cultures, nucleic acid amplification or direct antigen detection. Although these
methods are highly specific, their response can vary from 20 to 10 days using cell cultures. Rapid,
commercial influenza diagnostic tests are also available that have response times of 30 min. These tests
can provide results in a clinically relevant time frame to complement the use of antiviral medications for
treatment and chemoprophylaxis of influenza. Nevertheless, these systems have a low sensitivity (50-70 %)
and as a consequence, virus infected patients may go on undiagnosed until latter stages of the disease, where
treatment and cure are more difficult. In addition, undiagnosed patients are a source of dissemination of the
virus to others. The lack of sensitivity is a key limitation for using this kind of rapid test for Influenza
diagnostics.
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The proposed diagnostic system is an immunoassay based on a lateral flow test where virus detection is
done through the sensing of magnetic nanoparticles using state-of-the-art magnetoresistive (MR)
magnetic field transducers typically used in the read heads of the hard-disk drives. An integrated (microcoil)
inductive read out system will also be developed using high frequency magnetic field (tens of kHz) excitation.
The use of both approaches is justified because they are complementary, MR sensor is more suitable for small
dc magnetic fields and the inductive sensor allows for high ac exciting fields to be used.
Briefly, the system will work as follows: first the testing sample (nasal washes) containing virus is put in
contact with magnetic nanoparticles functionalised with an antigen particular to the particular virus strain. The
fluid in then put in contact with a porous membrane strip; the fluid sample migrates through the membrane
until the influenza virus + magnetic markers (if present) are recognized by membrane immobilized antibodies;
finally the presence of a particular virus strain is detected through the sensing of the magnetic flux created by
the bound magnetic nanoparticles, using a magnetic field transducer. This will enable a fast, specific and
sensitive diagnostic of the influenza virus, even if only present in minute amounts in the test samples.
Furthermore, patients will be diagnosed earlier, resulting in a more effective and efficient treatment and
prevent the spreading of the virus. Rapid detection of influenza virus is becoming increasingly important
given the concern for pandemic influenza caused by avian strains such as H5N1 or even, those engineered
strains that may be used in biological warfare. Thus, the proposed diagnostic system could also be suited for
the screening of large populations.
Core Project Aim and S&T Objectives
The proposed research will develop an integrated detection system for influenza virus strains. This will
include a lateral flow test for virus detection, a signal transduction device and an interpretation and data
analysis component. All modules of the system will be integrated within a single mobile system. The whole
system will be packaged as a single automated functional system with a simplified GUI interface. The aim
will be to deliver a customised product that targets for low to medium throughput, is low cost and can be used
for point-of-care applications. More importantly, the product will emphasize on providing very rapid and
accurate results.
S&T Workplan
The workplan for completion of the tasks and deliverables of the project is broken down into eight logical
workpackages as follows:
WP1: Development of immunoassays for influenza
WP2: Development of magnetic nanoparticles for influenza virus recognition
WP3: Development of a magnetoresistive based lateral flow test reader
WP4: Development of an induction based lateral flow test reader
WP5: Development of an easy-to-use control system that integrates the lateral flow test and reader devices
WP6: Market research and evaluation
WP7: Prototype biological assay testing/ System validation
WP8: Project management
The main project outputs of the project can be summarised as follows:
(1) development of influenza specific antibodies with optimized characteristics for lateral immuno assays
(2) development of surface functionalization protocols to immobilize influenza specific antibodies on porous
membranes ( lateral flow immuno assays) as well as on glass and smart stripes with incorporated electronics.
(3) development of magnetic nanoparticle markers functionalized with proper influenza
antigens/oligonucleotides
(4) development of a ultra high sensitivity (10pT/sqrt (Hz)) lateral flow magnetoresistive based stripe reader
A new generation of MgO based magnetic tunnel junction readers will be developed with sensitivities down
to 10pT/sqrt(Hz). These will be used to detect immobilized antibodies marked with magnetic nanoparticles.
The measurement of smaller virus concentrations. The magnetoresistive reader will be similar to a magnetic
card reader, with a mechanical device that will swipe in the functionalized stripe, read it out, and expel it.
(5) development of an integrated smart stripe (glass or plastic substrate with incorporated magnetopresistive
sensor, with immobilized antibodies on the surface, and microfluid channel for lateral flow tests)
Present prototypes developed at INESC MN and used for DNA-cDNA hibtridization detection have shown
the possibility of detection down to few tens ftmole/cm2.
(6) development of an inductive stripe read out system
The inductive sensor will be especially designed to detect the volume concentration of magnetic
nanoparticles. Target sensitivities are below 10pT/sqrt(Hz).
(7) development of the read out system mechanic/microfluidic interface, control electronics and user interface
for the three sensing platforms described in 4-6. The read out system mechanical/microfluidic interface will
be designed to allow user simplicity and reliability The control electronics will include a graphic user
interface, and the proper software, in order to allow assay control using an external PDA or laptop computer.
(8) demonstration of fully integrated and portable immunoassay platform prototype for rapid diagnostics of
influenza virus : comparison with existent fast detection kits. The key for the success of the project will be the
demonstration of higher sensitivity to influenza virus with the new detection platforms when compared with
existent fast detection kits ( 70% success).
(9) exploitation plan for the new devices based on magnetic nanoparticle markers
Protection of intellectual property concerned with the various phases of this project is critical to further
exploitation upon prototype tests. The coordinator will enforce IP protection strategies in this highly
competitive field. Market surveys of competitive technologies will be done throughout the project. It is
expected that the project will lead to a startup in biotech arena using highly sensitive antibody or DNA
screeners for particular applications.
Contribution to long-term innovation and the frontiers of knowledge
The proposed research will develop novel integrated, mobile, electronic platforms for the fast detection of the
influenza virus. It involves a multidisciplinary team bridging areas of nanotechnology, physics, electrical
engineering, and biochemistry, working together for a common goal: early detection of an infectious disease.
This type of platform, if successful, can and will be applied to a variety of medical situations, allowing point
of care diagnostics with a portable platform.
Potential socio-economic impact
The ability for European citizens to have easier access to fast diagnostics systems will have several socioeconomic benefits such as:
• Delays waiting for test results to come back from a central clinic will be avoided, thereby avoiding
the related stress and anxiety
• By providing a very accurate near realtime analysis system, errors and delays will be avoided,
thereby increasing the public confidence in genetics and biotechnology as a whole
• In the longer term, it will pave the way for point-of-care diagnostic systems that can provide
responses in relevant clinical time-frames, it can provide a valuable tool for screening biological
agents used in bioterrorism actions and furthermore, the technology can span to other fields requiring
fast analysis times such as for detection of pathogens in animals, food and beverages.
• Commercialisation of the system will provide new employment opportunities for highly skilled
graduates
Overall partnership
Scientifically, the overall partnership comprises four leading research institutes and three SME whose core
business are, respectively, products for immunochromatographic assays for clinical diagnostics and magnetic
particles for labelling of biomolecules.
Partner 1: INESC-MN, Lisbon, Portugal (RTD)
INESC MN will be the overall co-ordinator of the project (Prof. Paulo Freitas). INESC-MN will work with
the samples from partners to ensure that the sensor design and specification ensure optimal performance with
respect to signal-to-noise ratio, sensitivity and dynamic range using the specific bead labels proposed.
INESC-MN has already developed a magnetic sensor platform for detection of DNA hybridization and has
recently filed a patent application for the associated novel concept. INESC MN is one of the word leading
laboratories in the development of magnetoresistive biochips.
Partner 2: Certest Biotec, Spain (SME)
Immuno-chromatographic assay development using magnetic nanoparticles. Certes will be strongly involved
in the development of Influenza virus antibodies adapted for lateral flow tests (including magnetic particle
attachment). Certest will be responsible for workpackages 1 and 7, respectively design and fabrication of a
lateral flow test for Influenza virus detection, and testing of the integrated system (KIT). Certest is a Spanish
SME working in immunoassay diagnostic kits.
Partner 3: expert in Influenza virus antibodies (RTD)
Working with CERTES
Partner 4: Micromod Partikeltechnologie GmbH, Germany. (SME)
Micromod will adapt the surface chemistry of 20-30 nm diameter magnetic particles to enable direct
attachment of Influenza specific antibodies. Micromod has been working with INESC MN supplying various
types of functionalized magnetic nanoparticles for various biochip applications (Streptavidin coated, IgC
coated). Micromod is a German SME working in magnetic nanoparticle systhesis and functionalization for the
biomedical and pharmaceutical market.
Partner 5: Universidad de Zaragoza, Spain (RTD)
The group from the U. of Zaragossa is responsible for the design and fabrication of the inductive sensor
(WP4). This group has experience in inductive technology applied to AC susceptibility measurements and
will now apply its knowledge on the design and fabrication of a coil detector plus required signal
amplification electronics. This group has been working with Certest supplying magnetic nanoparticles and
helping Certes with SQUID magnetometry to quantify immunoassays using magnetic nanoparticles.
Therefore, it will also collaborate with CERTEST in the functionalization of the magnetic nanoparticles
produced in workpackage 2 and their magnetic characterization. Furthermore it will collaborate with INESMN in workpackage 3, participating in some of the fabrication process steps of an eventual new stripe with
integrated MR sensor. Finally it will be also responsible for the system integration (WP5) and system
evaluation (WP7) with respect to the inductive sensor performance.
Partner 6: INESC ID, Lisbon, Portugal
INESC ID will be in charge of task 6 (sensor integration), designing and manufacturing the system prototype
read out and control electronics. INESC ID has been working with INESC MN on the design and fabrication
of the signal acquisition, control electronics, and user interface modules for the spintronic biochips developed
at INESC MN. First results of this collaboration consist in the design and fabrication of the process control
module for a 256 biochip array involving thin film diodes and magnetic tunnel junctions.
Partner 7: Industry
Manufacture of the inductive sensor and the system read out and control electronics. Collaboration with
INESC ID and INA.