Final Report Summary - ASSAY FOR BIOMARKERS (Magnetic nanoparticles for multiplexed assays for low and high molecular weight biomarkers)
The application for the FP7-PEOPLE-2009-IEF was to support a move of a postdoctoral researcher to University College Dublin (UCD). The researcher as the time of moving had published 16 peer-reviewed journals with an h-index of 8. With the expert training and opportunities in place at UCD, the award of the fellowship hoped to build upon the successful start to his academic career and deliver him to a full time academic position. Upon completion of the proposal the fellow leaves with 22 publications, with cover 240 citations and has seen his h-index increase to 9. The fellow has been involved with three patent applications, and leaves the project to join the Chemistry Department at the University of Loughborough taking up a lectureship position.
The research proposal outlined a technique for the detection of biomarkers with the capability of working directly in biological samples. The technique aimed to deliver nanoparticles that contained paramagnetic and optical properties, which could be functionalised with aptamers / or antibodies. These functionalised particles would capture the biomarkers directly from solution, and in conjunction with an in-house technology named non-linear magnetophoretic (NLM) separation would allow the quick separation and detection by exploiting the particles superparamagnetic and optical properties, creating a new diagnostic technology. In developing the research it was apparent that the in-house NLM technology was not suitable for the detection of the analytes at this time and required further development. The fellow has contributed to the enhancement of the technology yielding publication in preparation. To circumvent the need of a detection platform a second technology was identified. A new emerging technology of tunable nanopore was utilised and has delivered a patent application, publications and commercial interest. Whilst the technology platform has changed from the initial proposal the ultimate aim has been secured.
The ultimate deliverable was a process capable of capturing the analyte as well as performing the initial sample purification and pre-concentration stage that screens multiple biomarkers for several diseases across a wide range of molecular weights and functionalities. Delivering the benefits of being both a multiplexed technique with the highly desirable tagless detection mechanism. The fellow has researched the key parameters for such assays as well as a new technology for this assay using nanopore technology. It has been demonstrated with aptamer capture probes and biomarker targets. The work has been showcased at two international conferences and has been the subject of commercial interest.
The training that accompanied this proposal aimed to ensure the fellow received a broad scientific understanding, crossing several scientific fields delivering an interdisciplinary research approach. When combined with the support infrastructure at UCD, it has delivered outstanding career enhancement prospects to the applicant, delivering a new diagnostic technique to enhance the European research portfolio. The applicant has been a mentor to two Doctor of Philosophy (PhD) students and has participated in teaching chemistry to undergraduate students.
Summary description of project context and objectives
Introduction
We deliver a new powerful diagnostic technique, built upon advances in the fields of separation and nanoparticle technologies. The scope of this technique and its far reaching implications will be demonstrated by the detection of large proteins and peptides alongside the detection of a small organic molecule. This allows the simultaneous detection of biomarkers covering multiple omic fields of research. The range of analytes that this technique is capable of detecting is made possible by developing new aptamer tagless detection strategies which will be incorporated for the first time onto nanoparticles. This approach will set the technique aside and beyond existing methods by combining several experimental aspects into one process. It has the capability to capture of the analyte, as well as perform sample preparation and extraction using magnetic particles, coupled with a fast and sensitive detection mechanism using both flow cytometry and nanopore technologies. This makes the outlined technique both simpler than existing alternative techniques such as mass spectroscopy or array based technologies, but it also produces a low tech miniaturised diagnostic device that has the capacity to be used in developing countries.
Microarrays are the dominant format for screening larger numbers of targets simultaneously, and are capable of yielding huge quantities of data at both the genomic and the proteomic level. Whilst there currently exist several commercially available antibody arrays, capable of screening for cell pathways or signalling sets, an equivalent format capable of screening metabolites is currently sought. Aptamers offer an alternative technology to antibodies, and since they were first raised in the 1990s, researchers have been quick to exploit their potential, with applications in drug candidate validation, therapeutics and diagnostics. An array combining both antibodies and aptamers would allow the screening of a large range of biomarkers spanning a range of molecular weights and functionalities. A limiting factor in creating such an array lies in the fact detection methods typically rely upon the analytes to be labelled / tagged using either a dye, enzyme or redox centre. Apart from introducing an additional experimental step and costs, it can be tedious and difficult to do for high throughput applications. In addition to this the label itself can often alter the method and strength of the biomolecular interaction leading to false positive of reduced sensitivity.
Metallic rods with nanoscale dimensions have been shown to be ideal reporters, sensors and diagnostic delivery agents. Our outlined fabrication process generates large quantities of particles with uniform and desirable characteristics. Huge efforts are underway to provide tagless detection mechanisms in diagnostic devices. Our proposed studies, provide a new route for tagless detection, highly publishable in itself, but would also provide a much sought after multiplexed assay.
We demonstrate this technology using technique with a variety of biomarkers with known antibodies and aptamers, once proven this technique will be quickly and easily adaptable to multiple systems as researchers discover new biomarkers for a range of diseases. With the use of aptamers alongside this technology and their growing body of research demonstrating the ease of modification and development of sequences. It is evident that if a tagless detection platform can be created, aptamer technologies can deliver a means to capture a diverse range of analytes.
Initially we use spherical beads and the flow cytometry technology to probe the kinetics and key parameters for particle aggregation and define the key parameters for tagless detection. We conclude with a new nanopore technology using multicomponent nanorods.
The research proposal outlined a technique for the detection of biomarkers with the capability of working directly in biological samples. The technique aimed to deliver nanoparticles that contained paramagnetic and optical properties, which could be functionalised with aptamers / or antibodies. These functionalised particles would capture the biomarkers directly from solution, and in conjunction with an in-house technology named non-linear magnetophoretic (NLM) separation would allow the quick separation and detection by exploiting the particles superparamagnetic and optical properties, creating a new diagnostic technology. In developing the research it was apparent that the in-house NLM technology was not suitable for the detection of the analytes at this time and required further development. The fellow has contributed to the enhancement of the technology yielding publication in preparation. To circumvent the need of a detection platform a second technology was identified. A new emerging technology of tunable nanopore was utilised and has delivered a patent application, publications and commercial interest. Whilst the technology platform has changed from the initial proposal the ultimate aim has been secured.
The ultimate deliverable was a process capable of capturing the analyte as well as performing the initial sample purification and pre-concentration stage that screens multiple biomarkers for several diseases across a wide range of molecular weights and functionalities. Delivering the benefits of being both a multiplexed technique with the highly desirable tagless detection mechanism. The fellow has researched the key parameters for such assays as well as a new technology for this assay using nanopore technology. It has been demonstrated with aptamer capture probes and biomarker targets. The work has been showcased at two international conferences and has been the subject of commercial interest.
The training that accompanied this proposal aimed to ensure the fellow received a broad scientific understanding, crossing several scientific fields delivering an interdisciplinary research approach. When combined with the support infrastructure at UCD, it has delivered outstanding career enhancement prospects to the applicant, delivering a new diagnostic technique to enhance the European research portfolio. The applicant has been a mentor to two Doctor of Philosophy (PhD) students and has participated in teaching chemistry to undergraduate students.
Summary description of project context and objectives
Introduction
We deliver a new powerful diagnostic technique, built upon advances in the fields of separation and nanoparticle technologies. The scope of this technique and its far reaching implications will be demonstrated by the detection of large proteins and peptides alongside the detection of a small organic molecule. This allows the simultaneous detection of biomarkers covering multiple omic fields of research. The range of analytes that this technique is capable of detecting is made possible by developing new aptamer tagless detection strategies which will be incorporated for the first time onto nanoparticles. This approach will set the technique aside and beyond existing methods by combining several experimental aspects into one process. It has the capability to capture of the analyte, as well as perform sample preparation and extraction using magnetic particles, coupled with a fast and sensitive detection mechanism using both flow cytometry and nanopore technologies. This makes the outlined technique both simpler than existing alternative techniques such as mass spectroscopy or array based technologies, but it also produces a low tech miniaturised diagnostic device that has the capacity to be used in developing countries.
Microarrays are the dominant format for screening larger numbers of targets simultaneously, and are capable of yielding huge quantities of data at both the genomic and the proteomic level. Whilst there currently exist several commercially available antibody arrays, capable of screening for cell pathways or signalling sets, an equivalent format capable of screening metabolites is currently sought. Aptamers offer an alternative technology to antibodies, and since they were first raised in the 1990s, researchers have been quick to exploit their potential, with applications in drug candidate validation, therapeutics and diagnostics. An array combining both antibodies and aptamers would allow the screening of a large range of biomarkers spanning a range of molecular weights and functionalities. A limiting factor in creating such an array lies in the fact detection methods typically rely upon the analytes to be labelled / tagged using either a dye, enzyme or redox centre. Apart from introducing an additional experimental step and costs, it can be tedious and difficult to do for high throughput applications. In addition to this the label itself can often alter the method and strength of the biomolecular interaction leading to false positive of reduced sensitivity.
Metallic rods with nanoscale dimensions have been shown to be ideal reporters, sensors and diagnostic delivery agents. Our outlined fabrication process generates large quantities of particles with uniform and desirable characteristics. Huge efforts are underway to provide tagless detection mechanisms in diagnostic devices. Our proposed studies, provide a new route for tagless detection, highly publishable in itself, but would also provide a much sought after multiplexed assay.
We demonstrate this technology using technique with a variety of biomarkers with known antibodies and aptamers, once proven this technique will be quickly and easily adaptable to multiple systems as researchers discover new biomarkers for a range of diseases. With the use of aptamers alongside this technology and their growing body of research demonstrating the ease of modification and development of sequences. It is evident that if a tagless detection platform can be created, aptamer technologies can deliver a means to capture a diverse range of analytes.
Initially we use spherical beads and the flow cytometry technology to probe the kinetics and key parameters for particle aggregation and define the key parameters for tagless detection. We conclude with a new nanopore technology using multicomponent nanorods.
