(Invited) Demonstration of ATPS Phase Separation, B-Field Concentration and Manipulation of Sars-Cov-2 Viral Particles

Abstract

A technology, based on fluorescence sensing in a liquid sample and capable for an early detection of a pathogen at extremely low concentration, is very desirable though equally challenging to develop. In optical sensors, a typical fluorescent signal would be too weak to measure without special tools, unless the detected analyte, tagged with a fluorescent label, can be separated and concentrated.In this work a new combined technology[1] will be presented. An assembly of a monoclonal antibody (mAb) functionalized with a fluorescent tag with the S-proteins at the surface of inactivated coronavirus is achieved. In order to separate a bound fluorescent tag (signal) and free tag molecules (background), we simultaneously attach to the virus surface ferrite magnetic nanoparticles (mNP) functionalized with the mAb (without fluorescent tag). Since the surface of the SARS-CoV2 virus has a large number of binding sites, a "sandwich" assembly of mNP-mAb:virus:mAb-FL happens. At the same time, non-specific binding of the fluorescent tag to mNPs is excluded. As a result, the fluorescent tag bound to the analyte can be manipulated by magnetic force and moved with respect to (unused) free tag molecules.Fig.1 shows the fluorescence signal of the mNP-mAb:virus:mAb-FL sandwich assemblies, that were first concentrated by an aqueous two-phase system separation (ATPS)[2] – carefully designed mix of PEG/dextran solutions. Upon dividing the ATPS into PEG bulk phase and dextran bubbles, the viral assemblies (and free non-bound tag) were separated into the PEG phase. Applying vertical magnetic field as shown in the figure resulted in additional significant concentration of the assemblies against the wall of a dextran bubble. Thus, we achieved a clear separation of the virus (as detected by direct fluorescence signal) with respect to the free tag molecules (background) residing in the bulk solution.[1] Vyacheslav V. Rotkin, Ming Zheng, Tetyana Ignatova, Daniel Hayes, Suresh Kuchipudi; Methods and systems for early detection of viral diseases. https://patents.google.com/patent/US20210364517A1[2] Albertsson, P. A., Partition of Cell Particles and Macromolecules. first ed.; Wiley: New York, 1986. Acknowledgement: This work has been supported by NSF (CHE-2032582 and CHE-2032601). Figure 1