Microplastic contamination

Approximately 400 million tonnes of plastic waste is produced globally every year. Although some of this waste is returned to plastic recycling plants, a large majority (~ 85%) ends up in urban landfills. Failure to effectively manage plastic waste has led to persistent environmental contamination. According to the UN Environment Programme, plastic waste entering aquatic ecosystems it is projected to reach 23-37 million tonnes per year by 2040 (UNEP, 2021).

Plastics are designed to be especially durable and resistant to degradation. As a result, plastic contaminants never fully break down, but erode to form microscopic plastic particles. Microplastics were estimated to make up 5 million tonnes of total plastic contamination in ecosystems in 2019 (UNEP, 2021). However, given the microscopic scale of these contaminants (less than 1mm) and a lack of routine monitoring, it is likely that this is an underestimate (Prata et al., 2019).

The bioaccumulation of microplastics in aquatic ecosystems is well established, but microplastics have also been found in human blood, lungs and placentas. One study estimated our intake of microplastics to reach up to 5 g/week (Senathirajah et al., 2021). Despite this, the effect of microplastic contamination on the environment and public health remains uncertain. Links have been made to antibiotic resistance and increases in water toxicity, and this has encouraged environmental agencies to propose far reaching restrictions on microplastic containing products. However, it has been emphasised that without routine monitoring efforts the risk that microplastic contamination poses will remain largely unaddressed (Prata et al., 2019).

Microplastic detection

There is a wide range of analytical approaches for the detection, quantification and characterisation of microplastics using microscopy and mass spectrometry methods. Fluorescence microscopy is commonly used for the initial inspection of microplastic samples with staining dyes, such as Nile Red. However, this does not provide insight into the chemical composition of the microplastics and quantification is unreliable. The chemical composition can be determined with spectroscopy methods, such as Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy, as well as mass spectrometry. Mass spectrometry has the advantage of analysing any toxic compounds that might be associated with the microplastics (Bauten et al., 2023). Nevertheless, there is no gold standard, as all the above approaches have their own challenges.

The absence of a universal protocol for microplastic detection has hindered studies aimed at assessing the global impact of microplastics. The more accessible fluorescence microscopy techniques lack uniformity and reproducibility. However, the alternative, using FTIR or mass spectrometry, is time consuming and expensive. To implement routine monitoring of microplastic contamination there is a need for simplified and accessible detection methods. Such methods should provide a level of quantification and insight into chemical composition, while remaining accessible (Prata et al., 2021). This is the challenge we aim to address as we develop our own microplastic biosensor.

Microplastic binding peptides

In recent years, novel methods for the detection of microplastics have focused on the use of peptide probes. These short amino acid sequences are able to bind with high specificity and affinity to different microplastics, such as polypropylene (PP) or polystyrene (PS). A multitude of these peptide sequences have been published with varying lengths, between 10 and 50 amino acids, and basic alpha-helical secondary structures (Woo et al., 2022). The success of these microplastic binding peptides (MBPs) has been reported in studies using fluorescence in microplastic detection. MBPs can be conjugated to fluorophores or fluorescent proteins, such as GFP (Bauten et al., 2023). Ji et al., developed an anchor peptide-GFP fusion protein that is capable of binding PP in solution allowing for visualisation (Ji et al., 2021).

The use of MBPs is a promising alternative for microplastic quantification and insight into chemical composition, given their specificity. Their success has also been proven in the detection of nanoplastics (less than 1 µm), which are notoriously difficult to detect and analyse below 100 nm (Oh et al., 2021). Despite the efficiency of using MBPs, these methods still rely on fluorescence microscopy and spectroscopy. Consequently, limiting their accessibility to those with the equipment and expertise necessary.

Engineering a bioluminescent biosensor

Bioluminescent enzymes have emerged as sensitive probes for in vivo imaging in recent decades. The enzyme luciferase catalyses the oxidation of the substrate luciferin resulting in light emission. Luciferase requires no external light stimulus, this resolving the issue of the background emissions that can decrease the sensitivity of fluorescent techniques. There is a wide variety of luciferase and luciferin pairs available, providing a range of signal intensities and colours (Yao et al., 2018). NanoLuc, taken from the marine shrimp Oplophorus gracilirostris, is one such luciferase that has been used as a biosensor in both medical and environmental contexts (Fig. 1). NanoLuc catalyses the oxidation of furimazine and is a popular choice amongst researchers given its thermostability and signal intensity (Zambito et al., 2021).

Figure 1: The light reaction catalysed by NanoLuc luciferase where furimazine is converted furimamide. The reaction produces an intense blue luminescence with a wavelength of aprroximately 460nm (England et al., 2016).

We aim to utilise the benefits of a NanoLuc biosensor for the detection of microplastics in water samples. Starting with the anchor peptide used by Ji et al., we have expressed our own fusion protein where GFP is replaced by NanoLuc luciferase. The fusion protein is deisgned to be used in a sandwich typed assay with the anchor peptide for detection and quantification (Fig. 2). The signal intensity of NanoLuc luciferase has promoted the development of smartphone applications for detection (Cevenini et al., 2016). With further development we aim to adapt this software for our own purposes in microplastic quantification. The success that we have achieved with our fusion protein promises to pave the way for an accessible and cost effective technology for microplastic detection in water samples.

Figure 2: A stylised version of our sandwich-typed detection assay. 1) Sample collection. 2) Incubation of microplastic sample with anchor peptide (orange) coated magnetic beads. 3) Microplastics bind to magnetic beads. 4) Addition of our NLuc fusion protein, the anchor peptide is in orange and NLuc in blue. 5) Apply a magnetic field to the sample and wash to remove unbound NLuc fusion proteins. 6) Add NLuc substrate furimazine. 7) Wait 5 minutes. The microplastic in blue is sandwiched between the two anchor peptides in orange. Note that one of the anchor peptides is also bound to a solid surface, 8) Detect the luminescent signal with your smartphone and use imaging software to analyse differences in signal intensity and quantify the microplastics in the sample.

References

Bauten, W., Nöth, M., Kurkina, T., Contreras, F., Ji, Y., Desmet, C., ... & Schwaneberg, U. (2023). Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput. Science of The Total Environment, 860, 160450.

Cevenini, L., Calabretta, M. M., Lopreside, A., Tarantino, G., Tassoni, A., Ferri, M., ... & Michelini, E. (2016). Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity. Analytical and bioanalytical chemistry, 408, 8859-8868.

England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjugate chemistry, 27(5), 1175-1187.

Ji, Y., Lu, Y., Puetz, H., & Schwaneberg, U. (2021). Anchor peptides promote degradation of mixed plastics for recycling. In Methods in Enzymology (Vol. 648, pp. 271-292). Academic Press.

Oh, S., Hur, H., Kim, Y., Shin, S., Woo, H., Choi, J., & Lee, H. H. (2021). Peptide specific nanoplastic detection based on sandwich typed localized surface plasmon resonance. Nanomaterials, 11(11), 2887.

Prata, J. C., da Costa, J. P., Duarte, A. C., & Rocha-Santos, T. (2019). Methods for sampling and detection of microplastics in water and sediment: a critical review. TrAC Trends in Analytical Chemistry, 110, 150-159.

Senathirajah, K., Attwood, S., Bhagwat, G., Carbery, M., Wilson, S., & Palanisami, T. (2021). Estimation of the mass of microplastics ingested–A pivotal first step towards human health risk assessment. Journal of Hazardous Materials, 404, 124004.

UNEP. (2021). From Pollution to Solution.

Woo, H., Kang, S. H., Kwon, Y., Choi, Y., Kim, J., Ha, D. H., ... & Choi, J. (2022). Sensitive and specific capture of polystyrene and polypropylene microplastics using engineered peptide biosensors. RSC advances, 12(13), 7680-7688.

Yao, Z., Zhang, B. S., & Prescher, J. A. (2018). Advances in bioluminescence imaging: new probes from old recipes. Current opinion in chemical biology, 45, 148-156.

Zambito, G., Chawda, C., & Mezzanotte, L. (2021). Emerging tools for bioluminescence imaging. Current Opinion in Chemical Biology, 63, 86-94.