Project Inspiration
As industrialization gradually spreads worldwide, more and more plastic wastes are mass produced each year to fulfill the demands of the public. It is expected for global annual plastic production to reach a shocking 1.1 billion tonnes in 2050 [1], proving that the massive production and consumption of plastic in modern life is causing disastrous consequences. Within all negative impacts, the most striking problem is obviously marine pollution.
Microplastic are plastic debris less than five millimeters in length [2]. They are commonly found accumulated in the ocean due to the degradation of plastics into smaller fragments in marine environments, insufficient sewage treatment due to incorrect disposal of plastic wastes, microplastic particles in cosmetic products [3] and fibers like polyester and polyamide [4]. After being consumed by marine animals, the plastic debris enters the food chain and bioaccumulates. Eventually, these microplastics enter our bodies as we consume goods from the ocean. Microplastics are toxic and harmful to the human body as they may cause health impacts such as oxidative stress, altering of metabolism [5], and pose of harm to gut microbiome [6], showing potential danger to both humans and marine creatures. The microplastics pollution problem is a great pain in the neck for humans.
Identification of microplastics in complex environmental matrices remains a challenge. Fast and accurate detection of microplastics in environmental water samples is essential for understanding their sources, occurrences and ecological concern. However, conventional detection methods require chemical or physical analytical techniques that are time-consuming and expensive. In Hong Kong, the detection method involved sample collection of 1000 L seawater and 500 L lake water by Manta trawling followed by sample pretreatment. Identification was done by counting visually under a stereomicroscope [7]. The procedural charge, equipments and shipping cost was expensive and therefore regular monitoring was impossible. Besides, the method for monitoring and quantifying microplastics in marine water varied from different countries making inconsistency in the global comparison [7].
Whole-cell biosensors, sensors that are formed from bacterial cells, can be modified by genetic engineering methods so that they can be used to detect a wider range of substances within a living cell. Due to their good sensitivity and high selectivity, they have emerged as tools for detection in the field of environmental monitoring, food analysis, pharmacology and drug screening [8]. After numerous background research and literature reviews, we found that the engineering of whole-cell biosensors for detection of microplastic level is unexplored. Also, there is no standardized testing protocol available for fast and accurate detection of microplastics and global comparison of pollution status. Thus, in this study, we engineered a whole-cell biosensor that expressed lacZ gene based on LasI-LasR quorum sensing regulatory circuit in P. aeruginosa. The detection and quantification of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal) solution act as a visible readout for the amount of quorum sensing signalling molecules, AHLs which ultimately can be used for rapid, sensitive and quantitative detection of microplastics. We aimed to develop a hydrogel-based biosensor to provide an alternative and standardized approach for microplastic detection and comparison of microplastic pollution in marine waters around the world.
Quorum Sensing System in P. aeruginosa
Pseudomonas aeruginosa (or P. aeruginosa), is a type of gram-negative, rod-shaped bacterium that can cause diseases in humans, such as infections in lungs in people who have cystic fibrosis, declined pulmonary function and others. It uses the quorum sensing system to control and produce virulence factors that are transcribed from the bacteria. P. aeruginosa contains a quorum sensing system LuxI/LuxR circuit. The Luxl homolog LasI synthesizes the signal molecule (AHL) 3-ox-C12-homoserine lactone (3OC12-HSL). The signalling molecule AHL is detected by the cytoplasmic LuxR homolog LasR. The LasR-AHL complex formed activates the transcription of target genes [9].
Quorum Sensing in Biofilms
Microplastics are plastics that have been shattered by various weathering processes or fabricated into small sizes for specific purposes with diameters of 5mm or less. Due to the hydrophobicity, large surface area and the pit-forming nature of microplastics, microplastics are known as carriers for microbial pathogens. Pathogenic bacteria have a high tendency to form biofilms on the surface of microplastics. This leads to an increase in the transcription of virulence factors within the bacterium due to a mechanism called quorum sensing by horizontal gene transfer that contributes to the increasing antimicrobial resistance (AMR) of pathogens [10].
The formation of biofilm is mainly controlled by the quorum sensing mechanism. Quorum sensing (QS) genes expressed as a result of the accumulation of a critical cell density and is involved in further cell to cell adhesion, maturation and dispersion of biofilm [10].
It is a cell-to-cell communication method between bacterium in biofilms. P. aeruginosa is a common Pseudomonas species that can attach to the microplastics and contribute to microplastic-associated AMR. When a colony of P. aeruginosa finds a piece of microplastic in the ocean, they will occupy it due to its smooth surface and numerous other factors such as high hydrophobicity [10]. They send out N-acyl-homoserine lactones (AHL) molecules, which are signaling molecules of P. aeruginosa. These signaling molecules attract more P. aeruginosa onto the piece of microplastic and forms a biofilm [11].
Conventional methods for microplastic detection
Conventional detection methods requiring chemical or physical analytical techniques that are time-consuming, expensive and required specialized equipments and expertise training. Examples including the use of Fourier transform infrared spectroscopy (FTIR) and Raman microspectroscopy which when used in tandem, could raise the problem of false negative [12].
In contrast, whole-cell biosensors can provide real-time, rapid and cost-effective detection for a broad range of environmental pollutants. Most of the host organisms encoding the sensor circuits are cheap and easy to handle compared to expensive and sophisticated equipment. Besides, the host cells can be genetically modified to detect a wider range of substances within a living cell with high sensitivity and selectivity. For example, a whole-cell biosensor expresses the transcription factor QscR sensing module detects water contamination by bacterial pathogen [13]. Genetically engineered whole cells can also be integrated into various solid supports to achieve portable biosensors [14].
Engineered whole-cell biosensors with implemented quorum sensing circuit represent a good alternative for environmental monitoring of microplastics pollution. Therefore, we aimed to apply P. aeruginosa LasI-LasR circuit to engineer biosensor for the detection of specific AHL molecules. The biosensor will ultimately use to quantitatively measure the presence of AHL molecules in water samples for monitoring microplastic pollution.
Design of the whole-cell biosensor
The whole-cell biosensor consists of two modules, the sensing module and the reporting module. The sensing module expresses the transcription factor LasR which can bind to the AHL molecule that are secreted from P. aeruginosa. Then the formation of LasR-AHL complex can bind to an inducible promoter, pLasRL or pLasR3 which lead to the activation of the reporting module. This whole cell biosensor contains a lacZ gene as the reporter gene, encoding β-galactosidase. The detection and quantification of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal) solution act as a visible readout for the characterization and optimization of the whole-cell biosensor in response to the quorum-sensing molecules of the P. aeruginosa, AHLs.
Figure 1. Schematic of engineered whole-cell biosensor containing the LasR sensing module integrated with pLasRL-LacZ reporting module. Abbreviation used are as follows: RBS, ribosome binding site; AHL, N-acyl-homoserine lactones.
Our Ultimate Goal
In this study, the highly efficient expression and easy detection of the blue color development of β-galactosidase activity was used to facilitate the characterization and optimization of the biosensor system. The engineered biosensor cells were physically immobilized in the alginate hydrogel and placed in the 96-well plate for blue colour development after a range of AHLs incubation. The protocol of AHL induction strategy and quantitative X-gal assay was optimized and standardized to develop a more sensitive method to detect even lower AHLs concentration. Ultimately, we aimed to develop the hydrogel-based biosensor which allows rapid, sensitive and quantitative detection of microplastics. It also provides an alternative and standardized approach for microplastic detection and comparison of microplastic pollution in marine waters around the world.
References
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