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Detection of Biomarkers using Nanorods and Nanopore technologies

Final Report Summary - NANO4BIO (Detection of Biomarkers using Nanorods and Nanopore technologies)

FP7-PEOPLE-2012-CIG 321836- Nano4Bio
Publishable summary
The application for the FP7-PEOPLE-2012-CIG was to establish a new research laboratory and advance the career of an academic starting his first position, whom at the time of submission published 22 peer reviewed journal articles and a h-index of 9 (using Web of science). The proposal expanded the applicant’s independent research investigating aspects of electrodeposition, particle synthesis, bio-functionalisation and particle translocation across a nanopore. It offered world class research with the opportunity to develop into a commercial technology providing Point of Care, POC, screening biomarkers.

At the end of the reporting period the applicants h index is 16 with 565 citations, and has published an additional 14 papers during the grant (with a further 4 submitted, and 2 in preparation at the time of writing), grown the research group to include five Ph.D students (two more to starting in Oct 2016). Three Graduate students have submitted their thesis (one already graduated, two defending in Dec 2016) one Research associate (with an additional RA to start in Oct 2016). We have supervised 6 students as MSc level graduates and one Erasmus student. The grant has provided the platform to grow the research, PI’s network now covering five countries, and the individual to a senior lecturer and a world leading researcher whose work has featured on the cover of Langmuir and Analytical Methods, within Biosensors and Bioelectronics facilitating collaborations with clinicians and industry.

Research aims: The research aimed to investigate a new nanotechnology combining particle synthesis with nanopore Coulter style devices, also known as Resistive Pulse Sensing, RPS. The purpose was to screen biomarkers directly from in biological samples. The synthesised particles contain a magnetic property creating superparamagnetic beads, SPBs. These particles provide the means to quickly concentrate a specific analyte from complex solutions with a high level of efficiency.

During this work we have pioneered the use of aptamers within tunable resistive pulse sensors, demonstrating the first tagless assays for thrombin using RPS technologies (Analytical Chemistry (2014) 86 (2) pp1030-1037). We have continued to develop the assay capabilities incorporating the first multiplexed assays on the TRPS platform for two cancer biomarkers (Biosensors and Bioelectronics (2015) 68, 741) and incorporated signal amplification and sample handling designed to be used at the bed side of the patient (Analytical Methods (2015)7, 8534-8538). To facilitate these assays we have developed new methods of analysis turning particle velocities into zeta potentials (Langmuir (2016) 32, 1082–1090.) and using them to characterise DNA modified nanomaterials, protein coronas around nanomaterials un serum and plasma (Anal Bioanal Chem (2016) 408: 5757) and the use of TRPS to study key biomarkers in patients of type 2 diabetes. The work will have a long legacy as it has facilitated collaborations with clinicians and the start of assays to screen blood sample for prions, and the development of a rapid assay to detect cardiovascular disease. The funding has facilitated the development of a multiomic, multiplexed bioassay technology, delivering world leading researcher that has enhanced the European union.

Summary of the objectives
Experimental objective 1 (4-39 months) Produce uniform particles with a magnetic functionality, and multiple component for surface chemistry.
Experimental objective 2 (10-33 months) Surface chemistry of Tri-component rods.
Experimental objective 3 (13-16 months) Enhanced sensitivity using ferromagnetic material.
Experimental objective 4 (10-34 months) Study of the effects of surface charge and orientation on translocation times.
Experimental objective 5 (10-48 months) Detect range biomarkers of molecular weights ranging from 1 kDa to 160 kDa.

Highlight clearly significant results
The move towards personalised healthcare is allowing biomarkers and disease to be measured and diagnosed without the need for centralised diagnostics laboratories. To facilitate the rapid and accurate diagnosis, it is desirable to perform several measurements at once in a multiplexed platform. In our work we describe a multiplexed assay for cancer biomarkerss PDGF and VEGF via tunable resistive pulse sensing (TRPS). Our method utilises aptamers, which are both easier to store and immobilise on surfaces and at a reduced cost when compared to antibodies. This is combined with a technology platform that requires no expensive optics, gas or fluid flow and a technology that can be easy integrated into microfuildic devices which is currently being manufactured on a large scale for DNA sequencing. By developing this methodology our assay could easily be implemented into POC testing.
This has been developed by studying the physical properties of particles surfaces, and understanding the double layer and zeta potential of DNA modified particles. In our work we have addressed and produced a theory of zeta potential measurements of charged particles, and their mobility in solution and through a nanopore. To date there are 7 papers based upon multiplexed detection using Resistive pulse sensors, this grant has funded two of these, and has one more stil be prepared. We have pioneered the use of aptamer nanomaterials and aptamer modified Tunable pores for the detection of biomarkers.
We have performed quantitative detection of small ions (Nanoscale 2016), multiplexed protein detection (Biosensors and Bioelectronics (2015) and provided new method of particle analysis using nanopore technologies based upon charge (Analytical Chemistry 2014, Langmuir 2016).