Exploring Fluorescence-Based Methods for Detection and Quantification of Model Viruses and Nanoplastics
Swarupa Chatterjee is a PhD student in the Department Nanobiophysics. (Co)Promotors ae prof.dr. M.M.A.E. Claessens and dr. C. Blum fron the Faculty of Science & Technology.
The aim of this research was to develop an alternative approach for the detection, quantification and sizing of weakly scattering nanoparticles (NPs) at (very) low concentrations in aqueous environment. This work presented a fluorescence-based methodology for the identification and quantification of two different categories of NPs: plant viruses (Cowpea Chlorotic Mottle Virus) as a model for the actual harmful pathogens and nanoplastics. Our approach involved labeling these NPs with specific fluorescent dyes and then using spectroscopic and microscopic techniques to detect them in aqueous environment. The advantage of using fluorescence-based detection is its sensitivity, versatility and moderate instrumental complexity. We have developed an instrument capable of detecting, quantifying and sizing the necessary low numbers of labeled model viruses and plastic particles, as well as methods for sampling the retention of labeled model viruses and detecting virus inactivation resulting from capsid disassembly by polymer-functionalised microfiltration membranes.
Chapter 1 provided a comprehensive overview of NPs, focusing on their applications, environmental impacts and the need for accurate detection and quantification methods. NPs have unique physical and chemical properties, such as enhanced reactivity and a high surface area to volume ratio, which are essential for advancing research and applications in various fields. The chapter identified these NPs as naturally occurring, accidental and engineered/man-made types, each with different origins and applications. The release of man-made NPs into the environment, mainly due to human activities, was highlighted as an environmental and health challenge. This chapter discussed the potential health effects of NPs, including their potential toxicity in organisms. It also explored the complexities of detecting and quantifying NPs in the environment, highlighting the limitations of current methodologies. Particular attention was given to viral NPs, especially waterborne viruses that are harmful to human health, and nanoplastics, which are products of the mechanical fragmentation of larger plastics. The chapter set the stage for further detailed quantification of these NPs, which require advanced detection and/or filtration methods. This quantification of viruses and nanoplastics would ultimately help to reduce their impact on human health and the environment.
Chapter 2 detailed the design and characterisation of single particle counting and tracking (SPC and SPT) for NP detection and sizing in aqueous suspensions, using fluorescent nanobeads as model particles. The setup, centred around a Nikon TE-2000U epi-fluorescence microscope, used two 2.1W multimode laser diodes (red and green) for excitation and a high numerical aperture objective to enhance photon collection. This chapter discussed the critical role of fluorescence in achieving significant contrast against the background, the construction of a fluorescence video microscopy setup, and the image analysis process facilitated by the Trackpy algorithm. The ability of this setup to detect NPs at low concentrations was confirmed by results demonstrating its ability to accurately count and track fluorescent nanobeads, visualised as diffraction-limited spots, even at very low concentrations. The image analysis technique, which included intensity thresholding for precise NP identification, emphasised the efficiency of this setup in identifying and monitoring fluorescent NPs down to minimal concentrations. Size determination of NPs was achieved by single particle tracking, with results benchmarked against fluorescence correlation spectroscopy data. This chapter culminates by highlighting the potential of the video microscopy setup in environmental research for the analysis of NPs at low concentrations.
In Chapter 3, we first focused on quantifying viruses at low concentrations. We used fluorescently labeled cowpea chlorotic mottle virus (CCMV) as a model virus, investigating how labeling with increasing amounts of fluorophores affected brightness and particle detection. The methodology involved labeling CCMV with different fluorophore densities and analysing using various spectroscopic techniques. The results showed a nonlinear relationship between fluorophore density and brightness, with higher densities leading to self-quenching and reduced fluorescence. The study further explored disassembling virus capsids to understand the fluorophore interactions. This confirmed that the close proximity of fluorophores led to decreased emission due to quenching. The photophysical data indicated the presence of non-fluorescent fluorophore aggregates and energy transfer to these aggregates, contributing to fluorescence quenching. Summarizing, we found out that, at already a low number of fluorophores per virus, fluorophore self-quenching and formation of dark aggregates limited the brightness of labeled viruses. For practical applications, we demonstrated the potential of the single virus particle counting approach at the optimized fluorophore density using our wide-field fluorescence microscopy setup. The brightest fluorescence was observed at intermediate fluorophore densities, facilitating virus detection in concentrations as low as 2 fM. This capability is significant for applications like optimizing membrane filters for virus removal using fluorescently labeled viruses for testing the membranes. It was concluded in this chapter that balancing the fluorophore densities is critical to achieving optimal brightness to detect viral particles.
Further in Chapter 4, we looked at an innovative way of monitoring the disassembly of virus capsids using complex fluorophore interactions. Virus capsids, the protein shells of non-enveloped viruses, are essential for their function. Established methods for assessing capsid integrity are often complex and resource intensive. In this work, we introduced a simpler fluorescence-based approach. The method exploited the photophysical interplay between multiple fluorophores attached to the capsid proteins. As the capsid disassembled, the photophysical interactions between the fluorophores changed, which could then be detected by spectral monitoring. To demonstrate the potential, a surfactant, sodium dodecyl sulphate (SDS), was used to induce disassembly of fluorescently labeled CCMV. The results showed that the method was effective, but the range of fluorophore labeling densities suitable for this assay was limited. High labeling densities could potentially affect capsid stability, and the spectral shifts observed upon disassembly were subtle. Despite these challenges, the research provided a promising basis for the development of simple fluorescence-based techniques to study virus capsid disassembly, which could be valuable for virus removal and inactivation strategies using microfiltration techniques.
Subsequently, in Chapter 5, we placed the emphasis on the effectiveness of a polyethylenimine (PEI) coated polyethersulfone (PES) microfiltration membrane in removing and inactivating viruses, particularly our model CCMV. The study employed SPC and spectroscopic techniques to quantify virus removal and assess the disassembly of virus particles, indicating their inactivation. Results showed that while the membrane efficiently removed intact viruses (achieving a 2log removal rate), a significant fraction of inactivated, disassembled viruses passed through it. Most viruses retained on the membrane were found in a disassembled, inactivated state. The interaction between the virus particles and the PEI coating was predominantly electrostatic. This finding suggested the possibility of membrane regeneration by altering the pH to disrupt these interactions. The study also confirmed that the PEI coating not only captured the viruses but also contributed to their disassembly, thus inactivating them. These insights could be useful in developing effective virus removal strategies and functionalized surfaces for water treatment applications, particularly in providing clean drinking water free from infectious viruses. The approach simplified the monitoring of virus inactivation and retention, a key challenge in developing new virus removal methods.
Moving forward, Chapter 6 marked a shift in focus to the detection and characterization of a second type of NP, namely, nanoplastics. We presented a method for detecting and quantifying nanoplastic particles as small as 45 nm. Utilizing a combination of fluorescence video microscopy and SPT with Nile Red staining, we successfully identified and measured nanoplastics in various suspensions, including those derived from commercially available polystyrene (PS) beads and ground disposable PS cups. The method was effective in distinguishing model PS particles of varying sizes, even in mixtures. It was able to accurately determine nanoplastic concentrations as low as 2×106 particles/ml. The approach involved staining plastic particles with Nile Red, which becomes strongly fluorescent upon adsorbing to hydrophobic surfaces like plastics. This enabled visualization and sizing of the particles through fluorescence microscopy. The size distributions of the particles were determined using SPT. The study also demonstrated the release of nanoplastics from a disposable PS cup into hot water, highlighting the practical applications of this method in real-world scenarios and highlighting how it may contribute to understanding the environmental impact of nanoplastics.
In Chapter 7, we addressed the challenge of detecting plastic particles smaller than 1 µm in real-world samples. This was challenging because the Nile Red used to fluorescently stain these plastic particles self-aggregated into particles in this size range. The presence of fluorescent Nile Red aggregates thus resulted in false positive counts. By optimizing Nile Red staining concentrations to nanomolar levels, we successfully reduced Nile Red aggregate formation while maintaining sufficient particle brightness for detection. For the experimental setting and the staining concentration used, we established a limit of detection (LOD) of 9 particles/nL and a limit of quantification (LOQ) of 30 particles/nL. Water samples of interest were subsequently stained with Nile Red and analyzed through fluorescence microscopy. In the quantification of nanoplastics in the samples, we accounted for Nile Red aggregates. Drinking water samples from plastic bottles and cartons showed approximately 250 particles/nL, significantly higher than tap water from Enschede, the Netherlands, which had a much lower nanoplastic concentration. The approach was also applicable directly to consumer-grade water samples without preanalytical treatment. This method offered a simple, cost-effective tool for accurate nanoplastic quantification in water, contributing to the understanding of environmental plastic pollution. Its low complexity and efficiency made it a promising technique for widespread use in nanoplastic research.
Finally, Chapter 8, provided insights into the remaining challenges and future prospects in the field of NP detection research, underscoring the continued need for innovation in this vital area.
