Fluorescence spectroscopy for the quantification of virus retention and inactivation efficiency of membrane filters

Harmful viruses in drinking water pose a threat to millions of people, especially in developing countries. One way to remove viruses from drinking water is by membrane filtration. A method to quantify virus retention and deactivation is mandatory to test, develop and optimise such membrane filters. Ideally, such a method is robust, fast and easy to implement. Currently used bio-assays-based methods to test the efficiency of membrane filters for the inactivation and removal of waterborne viruses are complex and time-consuming and, therefore, not well suited for membrane development, process optimisation and monitoring. Here we propose to develop an alternative method to follow virus retention and inactivation based on fluorescence. Our approach relies on fluorescent labelling of harmless (model or inactivated) viruses and spectroscopic detection of these viruses in the filtrate. Fluorescence-based detection is sensitive, versatile and of moderate instrumental complexity.

Goal

The proposed work focuses on identifying, counting and analysing the fluorescence from individual virus particles in the filtrate. Additionally, our labelling approach will allow the imaging of fluorescently labelled virus particles in the filter itself. Capsid disassembly and RNA/DNA release will be employed as readouts for virus inactivation and identification. In this way, we expect to be able to develop

• An instrument that can detect the necessary low numbers of labelled viruses;
• A method to sample the retention of labelled model viruses and mixtures of different viruses
• A method to detect virus inactivation due to virus capsid disruption or disassembly
• A method to identify native viruses in the filtrate based on DNA / RNA identification

Activities(scope), results and output

1. Design, build and validate instruments for virus detection and counting
2. Establish imaging of viruses in filters
3. Retention of labelled (model) viruses by membrane filters: (a) One model virus, (b) Virus mixtures, (c) Benchmarking, e.g. against MS2 phages
4. Virus inactivation by capsid disruption

Results

Molenaar, R., Chatterjee, S., Kamphuis, B., Segers-Nolten, I. M., Claessens, M. M., & Blum, C. (2021). Nanoplastic sizes and numbers: quantification by single particle tracking. Environmental Science: Nano, 8(3), 723-730. https://doi.org/10.1039/D0EN00951B

Chatterjee, S., Molenaar, R., Tromp, L., Wagterveld, R. M., Roesink, H. D., Cornelissen, J. J., … & Blum, C. (2021). Optimising fluorophore density for single virus counting: a photophysical approach. Methods and Applications in Fluorescence, 9(2), 025001. https://iopscience.iop.org/article/10.1088/2050-6120/abd8e4/meta

Chatterjee, S., Schotpoort, B. A., Elbert, T., Cornelissen, J. J., Claessens, M. M., & Blum, C. (2021). Exploiting complex fluorophore interactions to monitor virus capsid disassembly. Molecules, 26(19), 5750. https://doi.org/10.3390/molecules26195750

Chatterjee, S., Molenaar, R., De Vos, W. M., Roesink, H. D., Wagterveld, R. M., Cornelissen, J. J., … & Blum, C. (2022). Quantification of the Retention and Disassembly of Virus Particles by a PEI-Functionalized Microfiltration Membrane. ACS Applied Polymer Materials, 4(7), 5173-5179. https://doi.org/10.1021/acsapm.2c00560

Chatterjee, S., Krolis, E., Molenaar, R., Claessens, M., & Blum, C. (2023). Nile Red staining for nanoplastic quantification: Overcoming the challenge of false positive counts due to fluorescent aggregates. https://chemrxiv.org/engage/chemrxiv/article-details/645359361ca6101a45c17ba7