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Solid-phase synthesis of imprinted nanoparticles exhibiting glycan epitope recognition in glycoproteins

Periodic Reporting for period 1 - SSINGERG (Solid-phase synthesis of imprinted nanoparticles exhibiting glycan epitope recognition in glycoproteins)

Reporting period: 2019-10-01 to 2021-09-30

Protein glycosylation is potentially involved in all aspects of human growth and development. Aberrant/defective glycosylation of glycoprotein is undoubtedly a hallmark to diagnose all human diseases. Owing to its presence in low abundance, the effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. Molecularly imprinted materials (MIMs) technology has long held promises as capable of competing with antibodies by acting as chemically selective species in diagnostic assays, sensors and affinity separations/purifications. MIMs offer convincing advantages of low cost, high stability and do not require cold-chain logistics, contrary to biodegradable antibodies as it is expensive and required animals for preclinical treatment. Moreover, the synthetic nature of MIMs readily allows the incorporation of integrated signalling functionalities (colored, fluorescent, electroactive, etc.) during synthesis without affecting molecular recognition. Replacing it as an effective substitute for antibodies involving diagnostics or other health-related applications, MIMs must share key characteristics with their biomolecular counterparts, such as: aqueous solubility, size, affinity and selectivity for the target analyte. In order to satisfy all these criteria, it is important to prepare MIMs in the form of nanoparticles in a reproducible, controlled and scalable method. The nanoMIPs (nanoparticles of Molecularly Imprinted Polymers) produced by solid-phase synthesis, behaves as plastic antibodies, that is capable of selective discrimination and can be an ideal tool for glycoprotein enrichment. The present era is one in which diseases are constantly erupting, and researchers are baffled to obtain a quick and reliable technique to diagnose the illness. Since MIMs are proven to act as plastic antibodies, these materials can play a significant role in the selective and sensitive diagnostic of diseases. The present project was aimed at the preparation of synthetic nanoreceptors (nanoMIPs) which could be used as a better alternative in the efficient glycoprotein enrichment methods. Such nanoreceptors, behaving like antibodies, may overcome the current limitations, provided the enrichment of specific glycan subclasses, within studies of the glycoproteome are achieved. The preparation of the receptors will proceed through solid-phase synthesis of imprinted polymer nanoparticles, with the potential to deliver entities with size, specificity and solubility characteristics comparable to antibodies. Monosaccharides widely found in glycoproteins (Man, GlcNAc, GalNAc and Neu5Ac) was successfully anchored onto glass beads and, subsequently, NanoMIPs were produced using cautiously chosen monomer mixture with initiator. Performance of nanoMIPs was then evaluated by dot-blot technique. Two of the synthesised nano-MIPs exhibited high selectivity for glycoprotein over non-glycoprotein. Although the results were promising, a more detailed and systematic performance study was to be initiated in future.
The project activities had been carried out in four concrete work packages (WP 1 to WP 4) coherent with 4 tasks. Work packages WP 5 and WP 6 corresponded to dissemination and public engagements, respectively. Task 1 comprised of the successful immobilisation of monosaccharides with qualitative and quantitative assessment, and it pivoted one conference publication and also one Journal manuscript to be submitted soon.
Unforeseen COVID -19 crisis forced us to alter, postpone/ re-schedule some of the following project activities, WP 2 to WP 6 which included the secondment training at MIP-Diagnostics Ltd, UK. Although, WP 2 suffered a major impact, Dr.PK was able to synthesize 14 different nanoMIPs using free radical polymerization. The particle size data obtained by DLS and AFM proved it to be in the expected nanosized range. Data Management Plan was submitted to the European Continuous Reporting Portal and was approved by Project Officer.
In WP 3, Dr.PK underwent non-academic secondment training at MIP-Diagnostics Ltd., in UK where she established a strong collaboration with well-known scientists and mastered skills to synthesise nano receptors (plastic antibodies) by solid-phase synthesis, which was our main focus. Collaboration established between UP and MIP-Dx (the first in synthesising NanoMips) resulted in planning a new project which focused on “Sulfated glycoprotein enrichment” and it was submitted to FCT. With the experience gained from MIP-Dx, Dr.PK established a new tie-up with an eminent group i3S, dealing with Glycobiology and Cancer diagnosis.
WP 4 addressed the assessment of the synthesized nanoMIPs by dot-blot technique regarding its selectivity. Although rapid test results were promising, detailed study is to be conducted in near future to improve the selectivity of the nanoMIPs for glycoproteins.
The results obtained as of now, enabled Dr.PK to present herself as a speaker in "European University Alliance for Global Health”, EUGLOH. With more upcoming experiments, Dr.PK looks forward to publish an article having high impact factor. The inter disciplinary knowledge gained through this project, enhanced Dr.PK to assist one candidate pursuing PhD and also to act as a skilled scientific advisor for a Bio-engineering project. Besides that, Dr.PK also submitted a project aimed to synthesise molecular imprints as TRAP and ZAP approach for SARS-CoV-2.
New template of interest (Neu5Ac) in glyco-proteomics has been introduced in the project. Dr.PK was concentrating on the simplistic, low cost and, most importantly, novel methods focusing on Environmental protection by using non-hazardous reagents. New application of the immobilised saccharide material to classify binding specificities of several lectins has also been introduced in this project. Dr.PK mastered new techniques that can be adopted to identify and diagnose the diseases using nanoreceptors, with the help of corporate experts during the secondment training period.
The COVID pandemic has compelled a dire need to have a more efficient tool to identify and diagnose the disease at the earliest so as to avoid spreading rapidly. NanoMIPs as MIMs are one of the most readily available technique used for the SARS-CoV-2 diagnostics. Since these selective materials are a promising diagnostic tool, in future under crucial circumstances where new diseases erupt, it would be a valuable addition in current armamentarium of medical practitioners. Much to this, Dr.PK gained diverse knowledge in novel synthetic routes of immobilisation and could participate and learn to produce SARS-Cov-2 nanoMIPs on commercial basis at MIP-Dx, UK.
Overall, the SSINGERG project funded by Marie Curie Widening Fellowship, pitched up Dr.PK to multiple levels. Dr.PK also exceled in resilience, effective communication, project drafting/presenting skills and gained academic knowledge to a greater extent in working with eminent scientists during the course of this project. Dr.PK became a stronger candidate to work in academic and corporate sectors.
Solid-phase synthesizer
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