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Analysis of protein corona formation on nanoparticles by DNA barcodes

Periodic Reporting for period 1 - CoronaTag (Analysis of protein corona formation on nanoparticles by DNA barcodes)

Reporting period: 2016-03-01 to 2018-02-28

The aim of this project is to characterize the molecular buildup of protein coronas on nanoparticles and to clarify the role of corona proteins in the aggregation of nanoparticles in complex matrices, using state-of-art DNA-assisted protein technologies. The focus of the project will be on nanoparticles relevant for diagnostic purposes, where molecular recognition is effectuated by affinity-reagents and the nanoparticles are used for target enrichment and signal generation.

The findings generated from the proposed project will be of great importance in developing nanoparticle-based biosensing technologies for POCT devices, which reduces the turn-around times to the physician, resulting in faster treatment decisions, giving improved workflows and improving the quality of care . The opportunities to perform testing in less expensive settings such as the doctor's office, home of patients, or even through remote control will improve the accessibility of care, reduce overall healthcare costs, and improve the personalization of treatment. Since 2010, the FP7 and H2020 calls for proposals have included personalised medicine as one of the research priorities and a number of topics have made specific reference to personalised medicine approaches, showing the importance attached to the area. With the help of TU/e Innovation Lab and the collaboration with Philips Research, the outcome of the findings can be further translated to potential industrial products, which will further benefit the European society.

The objectives of the action will be:
• Investigate protein corona formation on nanoparticles in parallel: The identity of proteins will be converted to DNA by tagging plasma proteins and affinity reagents with functional DNA tags (WP1).
• Investigate dynamics of the protein corona formation by mild crosslinking (WP2): The rapid exchange of molecules in the soft corona on the nanoparticles can be studied by crosslinking them to the hard corona and the nanoparticle surface at different time points.
• Corona proteins together with affinity reagents will be investigated in three biosensing configurations that are important in diagnostic assays (WP3): The interactions between affinity reagents and targets, between affinity reagents and corona proteins, and between different corona proteins, will be measured by ligated DNA tags.
The work performed is summerized as work packages below:
WP 1. Conjugating proteins with DNA tags
WP 1.1 Conjugation parameters: Albumin and IgG will be conjugated with DNA oligonucleotides with different lengths and numbers on the protein. Conjugates will be tested for stability and conformation changes by thermo shift assay and Circular dichroism compared with unconjugated proteins. Conjugates will be tested for proximity ligation functionality.
WP 1.2 Create DNA-barcoded library of corona proteins: A list of corona proteins such as albumin complement C3, fibronectin, transferrin, IgG, IgM etc and clinically relevant proteins such as PSA, and antibodies against PSA, will be labeled with DNA tags (both 3’free and 5’free) to create a DNA-barcoded protein library which will be used in WP 2 and 3. The protocol will follow what is collected from WP 1.1.
WP 2. Pilot study of corona formation in complex matrix
WP 2.1 Create partially labelled complex matrix: Complex matrices such as plasma will be depleted to remove endogenous proteins (albumin, IgG) and replace these by DNA-barcoded proteins prepared in WP 1, in order to create partially labelled plasma.
WP 2.2 Quantification of albumin and IgG within corona on nanoparticles: will be investigated using partially labelled plasma prepared in WP2.1 by proximity ligation and qPCR.
WP 3. Protein corona formation study
WP 3.1 on colloidal nanoparticles (cf. Fig. 3a) with analysis of proteins and protein colocalization in the corona on the nanoparticles
Progress is summerized as deliverables below:
D1: Conclusion on the optimal length and number of oligonucleotides per protein, establishment of protocols for conjugation;
D2: Preparation of DNA-barcoded protein library of corona proteins that can be used in WP2 and WP3
D3: Corona profile measured for albumin and IgG on nanoparticles, with qPCR and fluorescence microscopy
D4: Data collection of corona protein formation dynamics from qPCR or next-generation sequencing and analysis of the interplay between protein corona and affinity reagents
D5: Conference and workshop presentation: Study of protein corona on nanoparticles using DNA-assisted protein technology.
overview of the workflow of the project