Development of a Cellular Sensor Detecting Heavy Metals and Antibiotic Residues in Minute Quantities

Project Description

There are various contaminants in our environment.


Can heavy metals and antibiotics in environmental liquids be effectively detected?

Our aim is to detect these contaminants.

Our heavy metal biosensor

The basis for the detection of specific heavy metals is the metal-responsive transcription factor 1 (MTF-1). This protein is a nucleocytoplasmic shuttle protein that is able to bind certain heavy metals and subsequently migrates into the cell nucleus, where it binds to promoters containing metal-responsive elements (MRE). Thereby, expression of target genes can be regulated. In our sensor, we intend to utilize this system to express a fluorescent protein in order to make the presence of heavy metals visually apparent.[41]

Figure 3: Signaling pathway of the heavy metal biosensor
Figure 4: β-lactam detection circuit

Our antibiotic biosensor

To detect β-lactam antibiotics, we aim to utilize the protein PknB, a eukaryotic serine/threonine kinase orthologue from Staphylococcus aureus. In addition to an intracellular kinase domain, this protein has extracellular PASTA (for penicillin-binding protein [PBP] and serine/threonine kinase-associated) domains. The PASTA domains are capable of binding ß-lactam rings, which results in autophosphorylation of the protein and the subsequent activation of downstream signaling cascades[37]. PknB is able to phosphorylate both human and bacterial proteins, which increases the binding activity of the phosphorylated protein to the promoter region. To ensure the functionality of the sensor, we will introduce three distinct proteins, known to be phosphorylated by PknB into the cells and subsequently activate the expression of certain genes. These proteins are GraR and CcpA from S. aureus and the human ATF-2[40]. As with the metal detector, a fluorescent protein should indicate gene activation and thus make the presence of β-lactam antibiotics visible. 

Antibiotic and Metal Residues

In a world where invisible forces of contamination present a challenge to our health and environment, we must recognize and address antibiotic and metal residues as silent adversaries. These persistent pollutants, remnants of medical and industrial activities, seep into our water, food, and soil, and they are undermining the very ecosystems we rely on. We can change that! We can and must tackle antibiotic resistance head-on, protect aquatic life, and ensure the safety of our resources. Their presence, often undetectable by conventional means, presents an opportunity for us to do so. We are proud to present our revolutionary sensor, a vital tool in the fight against these hidden hazards. This cutting-edge solution is designed to detect antibiotic and metal residues with unparalleled accuracy, and it will be a game-changer in safeguarding public health and preserving the delicate balance of our environment. The future of a cleaner, safer world begins here!

A Significant Threat to Ecosystems and Human Health

A significant environmental crisis is unfolding beneath the surface of our daily lives. It is contaminating our rivers, lakes, and even the water we drink. The byproducts of modern agriculture, medicine, and industry are antibiotic and heavy metal residues. These contaminants are increasingly contaminating aquatic ecosystems and the environment that depends on these ecosystems. The contaminants primarily originate from pharmaceutical manufacturing, hospital and agricultural wastewater, and industrial runoff, but also form our own daily behavior. Once released, they do not degrade easily. This inevitably leads to their accumulation in the environment and their entry into the food chains of humans and other living organisms.
Next to their high toxicity, heavy metals are known to cause severe environmental and health problems with prolonged exposure, even at low concentrations. Heavy metals accumulate in living organisms through bioaccumulation instead of breaking down over time. Studies have shown that heavy metals affect cellular structures and interact with cell components, and that some heavy metals like lead, mercury, and cadmium are hazardous even at low levels.[1]
Antibiotics, essential for treating bacterial infections, have become major environmental contaminants due to their extensive use and potential improper disposal. When antibiotics enter natural water systems, they can have a big impact on the sensitive natural equilibrium. Microbial communities can be disrupted, and antibiotic-resistant bacteria fostered due to the presence, undermining the effectiveness of vital medical treatments. [2, 3, 4, 5]
We must address the role these contaminants play in the development of antibiotic resistance. There is no doubt that the presence of antibiotics in the environment exerts clear and decisive selective pressure on bacterial populations, promoting the survival of resistant strains. Furthermore, co-selection (co-resistance or cross-resistance) can occur when bacteria exposed to heavy metals develop resistance mechanisms that also provide resistance to antibiotics, thereby exacerbating the spread of multi-resistant pathogens. [6, 7]

There Is a Need for a New Kind of Sensor for These Contaminants

The current solutions, such as conventional filtration and water treatment methods, are insufficient for addressing this complex problem. Many wastewater treatment plants face challenges due to insufficient infrastructure for effectively removing antibiotic and heavy metal contaminants. This allows these pollutants to persist in the environment and continue to pose risks to both ecosystems and human health. [8] This highlights the urgent demand for sophisticated detection and remediation technologies, which can accurately identify and quantify these contaminants before they cause irreparable harm.
It is therefore essential to implement continuous monitoring and detection of both heavy metals and antibiotic residues in liquid samples in order to protect human health and the environment. Despite the existence of feasible methods, there is a necessity for the development of advanced technologies that can overcome the limitations of conventional approaches. In particular, this is true given that heavy metals and antibiotics are also known to directly interact with each other, causing co-selection and antibiotic-metal complexes (AMCs) with a bioactivity profile and physicochemical properties different to non-complexed residues. [9]
We are dedicated to pursuing innovative solutions that directly confront the challenges posed by antibiotic and metal residues. To address the contamination of water by antibiotics and heavy metals, we must adopt a multidisciplinary approach and drive innovation. Our sensor technology is a game-changing advancement in this effort. It provides the precise and reliable means of detecting these contaminants. By enabling early detection and accurate monitoring, we will better protect our ecosystems and public health from the long-term consequences of these pollutants.
We are thrilled to introduce the Hydro Guardians project, which will develop a revolutionary cellular sensor based on mammalian HEK cells. This cutting-edge technology will detect a range of heavy metals and antibiotic residues in liquid samples early. This incredible sensor is designed for extremely precise and fast detection. It also creates a synthetic-biological basis for the further development of previous detection approaches! The sensor uses genetically modified cells that convert residues into visual signals. These cells react to specific heavy metals or antibiotics, making them highly sensitive indicators. Based on our innovative concept, we are convinced that this sensor will significantly speed up detection and help us issue early warnings about harmful contaminants in food, drinking water, and the environment.

Sources of Water Pollution

The Unseen Perils: Diverse Sources of Water Pollution

The contamination of water resources represents a pervasive and multifaceted threat to the health of our planet's vital waterways. The sources of this contamination are as diverse as they are insidious, with antibiotics and heavy metals representing two of the most concerning pollutants. A comprehensive understanding of these sources is indispensable for the development of effective solutions to safeguard the environment.

Antibiotics in Water: A Growing Danger

The contamination of water with antibiotics has become a significant environmental concern, with a variety of potential sources. These include the pharmaceutical manufacturing, medical usage and waste, agricultural practices and household disposal and personal care products. The introduction of antibiotics into aquatic systems can disrupt the natural balance of ecosystems and pose a risk to human health.

  1. Pharmaceutical Manufacturing: The pharmaceutical industry is a significant contributor to water pollution, with antibiotic residues frequently detected in wastewater. It is not uncommon for residual antibiotics and by-products of manufacturing processes to enter wastewater streams. In many settings, particularly in regions with less stringent regulations, wastewater treatment may be inadequate, resulting in the direct release of these antibiotics into rivers and lakes, thereby directly affecting the aquatic life and environment in these regions. [5, 10]
  2. Medical Usage and Waste: The utilization of antibiotics in healthcare settings has been identified as a significant contributor to water pollution, largely due to patient excretion and inadequate disposal practices. Antibiotics excreted in urine and feces enter municipal sewage systems, where conventional treatment plants often prove incapable of removing them entirely due to limitations inherent to current technologies. The practice of flushing unused medications, for instance, represents an additional source of contamination, as these substances are introduced directly into the water supply. [5, 10, 11]
  3. Agricultural Practices: The agricultural sector is another significant source of water pollution. The use of antibiotics in livestock and aquaculture results in the presence of these pharmaceutical agents in manure tanks or waste, which can then enter water bodies such as groundwater or aquatic environments through leaching and overland flow runoff. Such contamination has an adverse effect on water quality and disrupts the natural ecosystem. [5, 12]
  4. Household Disposal and Personal Care Products: The inappropriate disposal of antibiotics and personal care products containing antibacterial agents also contributes to the contamination of water resources. A significant number of individuals dispose of unused medications by flushing them down toilets or sinks, which results in direct contamination of water bodies. The use of personal care products such as antibacterial soaps and sanitizers, which contain active antibiotic ingredients, can result in the further introduction of these compounds into wastewater systems. [5, 11] Based on the significance of this issue – and our all chance to address it – we decided to specifically to create a children’s book for educational purposes. This book and the story behind it can be found in the Human Practices section.

Heavy Metals in Water: An Industrial Legacy

Heavy metals are another major source of water pollution, with severe impacts on both water quality and ecosystem health. The contamination with heavy metals is the result of a number of significant sources like industrial discharges, mining operations, agricultural runoff and atmospheric deposition, each of which contributes to the pervasive presence of these toxic elements in aquatic environments.

  1. Industrial Discharges: Industrial discharges are a significant source of heavy metal pollution in water bodies, particularly from sectors such as mining, metallurgy and chemical manufacturing. These industries release toxic metals such as lead, mercury, cadmium and chromium into the environment through inadequately treated wastewater. The electronics and textile industries also contribute by discharging metals used in production processes. Inadequate wastewater treatment intensifies this problem, allowing harmful metals to accumulate in aquatic ecosystems. [1, 13]
  2. Mining Operations: During the extraction and processing of ores, mining activities expose and release heavy metals such as arsenic and mercury into the environment. These pollutants can persist in sediments and accumulate in the food chain, leading to long-term ecological and health risks. [1, 13, 14]
  3. Agricultural Runoff: The use of fertilizers and pesticides in agriculture introduces trace amounts of heavy metals into the final product and soil. These metals can then be washed off fields during rainfall and enter waterways. In addition, the use of sewage sludge as a fertilizer can also introduce heavy metals into soil and water systems. The aforementioned runoff can lead to the accumulation of metals such as cadmium and lead in aquatic environments. [1, 13, 15]
  4. Atmospheric Deposition: Heavy metals also enter aquatic systems through atmospheric deposition. Industrial emissions, vehicle exhaust, and the burning of fossil fuels release metals into the air, which are then deposited on land and water surfaces by precipitation, resulting in widespread contamination of terrestrial and aquatic ecosystems. [1, 13, 16]

By recognizing and addressing these diverse sources of water pollution, we can work toward more effective strategies for protecting our vital water resources.

Impact on the Environment

The Environmental Impact of Antibiotic and Metal Residues

The presence of antibiotic and metal residues in aquatic environments represents a significant environmental and public health concern, given their harmful impact on ecosystems and human health. These pollutants, which are commonly found in water bodies around the world, have the potential to cause significant disruption to the ecological balance, endanger biodiversity, and contribute to the growing issue of antibiotic resistance. The interaction between these pollutants amplifies their effects, rendering them particularly challenging to address.

Impact of Antibiotic Residues

The introduction of antibiotics into waterways can have unintended consequences, including the disruption of beneficial microbial communities that are involved in essential nutrient cycling processes. Even low concentrations, which are often found in wastewater, have the potential to alter microbial populations and disrupt ecosystem stability, which can result in adverse effects such as algal blooms and oxygen depletion. [17, 18, 19]
A significant concern is the emergence of antibiotic-resistant bacteria, which is facilitated by the selective pressure exerted by antibiotic residues. The transfer of resistance genes from antibiotic-resistant bacteria to other bacteria, including pathogens, has the potential to exacerbate the global antibiotic resistance crisis. [20, 21] Furthermore, the direct harm caused by antibiotics to aquatic organisms, such as fish and algae, can lead to a reduction in biodiversity and ecosystem productivity. [22, 23]

Impact of Metal Residues

Heavy metals have the potential to be highly toxic to aquatic life, with the capacity to affect enzyme function, respiration, and reproduction. Furthermore, they can induce oxidative stress by generating reactive oxygen species (ROS) within cells, which can result in cellular damage, inflammation, and apoptosis. This oxidative stress can have significant consequences for the health of aquatic organisms, including reduced growth rates, compromised immune systems, and increased mortality. [1]
Heavy metals can also bioaccumulate in the tissues of aquatic organisms, thereby biomagnifying up the food chain and posing significant risks to top predators and humans. Bioaccumulation occurs when organisms absorb metals at a rate that exceeds their capacity for elimination, resulting in an accumulation of these substances within the body over time. Beyond that, heavy metal contamination can result in long-term ecological damage, as top predators often serve as critical species in maintaining ecosystem balance. [24]

Combined Effects of Antibiotic and Metal Residues

The combined toxicity of antibiotics and metals is greater than the sum of their individual effects, resulting in more severe ecological damage. For example, studies have demonstrated that the presence of copper can enhance the uptake of antibiotics by bacteria, resulting in more pronounced disruptions in microbial communities and greater ecological damage. [6]
Furthermore, metals can facilitate the co-selection of antibiotic resistance genes in bacteria, leading to the emergence of multi-resistant bacteria and complicating the management of antibiotic resistance. The dissemination of these co-selected resistance genes represents a significant challenge to public health, as it complicates the control of antibiotic resistance and elevates the risk of infections in humans that are resistant to treatment. [6, 7]

Current Solutions

Current Solutions for Measuring Antibiotic and Metal Residues: The Challenges and the Need for Innovation

The presence of antibiotic and metal residues in the environment represents a significant public health and ecological concern, underscoring the urgent need for accurate and reliable detection methods.
Traditional chemical analysis techniques are highly sensitive and precise, enabling the detection of trace contaminants. Despite their precision, these techniques present significant challenges, including high costs, labor-intensive procedures, and the requirement for highly trained personnel. The complexity of sample preparation and the lengthy analysis times further complicate their use, making them less feasible for routine monitoring, especially in resource-constrained settings. [25, 26, 27, 29]
Spectroscopic and electrochemical methods often offer a balance between sensitivity and ease of use, making them suitable for routine analysis in controlled environments. However, they require extensive calibration, trained personnel and sophisticated equipment, which limits their application in resource-limited areas. [25, 28, 29]
Biosensors have emerged as a promising alternative for the detection of antibiotic and metal residues, offering faster and more user-friendly analysis through the use of biological components. While they allow for real-time analysis and can be more cost-effective, the sensitivity and specificity of biosensors vary widely. Nevertheless, they can have a high specificity and sensitivity with low detection limits. An easy-to-use application and a minimal sample preparation can lead to good usability in various non-laboratory environments. [25, 30, 31]
These advances must be combined with innovative solutions that combine the accuracy of traditional methods with the simplicity and cost-effectiveness of newer technologies. Current methods are scientifically robust but impractical for widespread use, especially in areas most affected by contamination. Developing such solutions is not only a scientific challenge but a moral imperative. It is crucial for enabling effective environmental monitoring and timely intervention to protect public health and ecosystems. [5, 8, 25, 29]

Threat to Human Life

The contamination of water by antibiotics and heavy metals poses a significant threat to human health and the environment. It undermines global efforts to achieve several United Nations Sustainable Development Goals (SDGs). These contaminants impact individual health, public health systems, ecosystems, and the long-term sustainability of natural resources. [32]

To find out how exactly our project can help achieve the SDGs, check out our Sustainability page!

Cross-Resistance between Metals and Antibiotics

The emergence and spread of antibiotic resistance is a major global health threat. While antibiotic use is considered to be the primary driver of resistance, there is growing evidence that exposure to metals can also contribute to antibiotic resistance through co-selection mechanisms[33, 34].

Mechanisms of Cross-Resistance

Cross-resistance between metals and antibiotics refers to a phenomenon where bacteria that develop resistance to heavy metals, such as copper, zinc, or mercury, also exhibit resistance to certain antibiotics, even without prior exposure to these antibiotics[34]. For metals and antibiotics, the principal mechanisms include:

  1. Co-Resistance
    Co-resistance involves the presence of resistant genes for both metals and antibiotics on the same mobile genetic elements, such as plasmids, transposons, or integrons. This genetic co-localization allows for the simultaneous acquisition and retention of resistance traits. For example, plasmids carrying genes for heavy metal resistance often also carry genes for antibiotic resistance, facilitating horizontal transfer of these genes between bacterial species. This process accelerates the spread of resistant genes across diverse environments and bacterial populations[34, 35].
  2. Cross-Resistance
    Cross-resistance occurs when a single resistance mechanism, such as an efflux pump, can expel both metals and antibiotics from bacterial cells. Efflux pumps can actively transport a wide range of substrates out of the cell, including both metals and antibiotics. This broad substrate specificity allows bacteria to develop high levels of resistance to multiple toxic agents simultaneously[33].
  3. Co-Regulation
    Metals can act as signaling molecules that modulate the expression of resistance genes through regulatory networks. For example, heavy metal ions can activate metal-responsive transcription factors, which in turn upregulate genes associated with both metal and antibiotic resistance. This co-regulation is often mediated by metal-induced regulatory proteins that affect gene expression at the transcriptional level. One example is the Metal-Responsive Transcription Factor 1 (MTF-1), which responds to metal exposure by binding to metal-responsive elements in the promoters of genes encoding both metal and antibiotic resistance[35, 34].
  4. Antibiotic-Metal Complexes (AMCs)
    Antibiotic-metal complexes (AMCs) form when metals interact with antibiotics, resulting in the formation of complexes with altered physicochemical properties. These complexes can significantly affect bacterial resistance mechanisms by altering the bioavailability and activity of antibiotics. For example, metals such as zinc or copper can bind to β-lactam antibiotics, potentially altering their efficacy and allowing bacteria to evade their effects. AMCs can also increase bacterial resistance by facilitating the uptake or stabilization of both metals and antibiotics in the microbial cell, complicating treatment strategies in environments contaminated with these complexes[34].

Tackling the Challenges of Cross-Resistance

Our project addresses the challenge of cross-resistance by integrating two advanced biological systems: Metal-Responsive Transcription Factor 1 (MTF-1) and the PknB protein kinase. MTF-1 is a key component in detecting metal-induced stress and regulating gene expression related to metal resistance, while PknB, a serine/threonine kinase from Staphylococcus aureus, facilitates the detection of β-lactam antibiotics through its PASTA domains. By incorporating these systems into our biosensor, we can monitor the presence of heavy metals, antibiotics and their interactions, including the formation of AMCs. This integration provides a comprehensive approach to understanding and mitigating the complexities of cross-resistance in contaminated environments.

The SynBio "Hydro Guardian"

Dual Sensing with Prokaryotic and Eukaryotic Components

Our team has developed an innovative cellular sensor to prevent the spread of heavy metals and antibiotics at an early stage and counteract the formation of resistance. This sensor is able to detect β-lactam antibiotics and heavy metal residues in water. We chose to base our sensor on HEK (Human Embryonic Kidney) cells due to their high transfection efficiency and proven ability to show fast and precise responses to external stimuli.

By integrating the antibiotic-detecting PASTA domain from Staphylococcus aureus and the metal-responsive transcription factor MTF-1, which is conserved in many organisms from insects to vertebrates, we hope that our cellular sensor can detect heavy metals and antibiotics at an early stage to effectively counter their spread.

Combining Antibiotic and Metal Detection

Developing a biosensor that can simultaneously detect antibiotics and heavy metals is crucial for addressing two major environmental and public health concerns. Antibiotic contamination in water and soil contributes to the rise of antibiotic-resistant bacteria, which poses a serious threat to human health. Meanwhile, heavy metal pollution from industrial activities can lead to toxic accumulation in ecosystems and food chains, causing adverse health effects. A dual-detection biosensor would enable comprehensive monitoring of these contaminants, facilitating timely interventions and promoting safer environments and healthier populations.

The PASTA Domain of Staphylococcus aureus

The PASTA (penicillin-binding-protein and serine/threonine kinase-associated) domain is an important structural component in certain proteins that is characterised by its ability to recognise and bind beta-lactam compounds.[36] Our approach uses the PASTA domains of the kinase PknB to construct a sensor that can detect the presence of antibiotics in the environment.

β-lactam detection circuit

PknB is a eukaryote-like serine/threonine kinase in Staphylococcus aureus that consists of an N-terminal cytosolic kinase domain, a central transmembrane domain and three C-terminal extracellular PASTA domains.[36] When this extracellular domain recognises beta-lactam compounds, it transmits the signal by autophosphorylating the N-terminal kinase domain, thereby activating downstream signalling cascades.[37] In S. aureus, this mechanism normally enables early recognition and adaptation to antibiotic stress.[38]

In our project, we introduce PknB into HEK cells, together with three proteins that can be phosphorylated by PknB. This targeted phosphorylation increases DNA binding activity [39] and induces the expression of a specific gene, which generates a fluorescent signal. To ensure the functionality of our sensor, we have chosen three different proteins that have been shown to be phosphorylated by PknB. These proteins are two transcription factors from S. aureus GraR and CcpA [39, 40], as well as the human transcriptional activator ATF-2.[41] To demonstrate antibiotic detection, we also designed a highly active promoter and introduced it into the cells, which enables us to measure the activation of gene expression by means of a fluorescent signal. 

MTF-1 is a Eukayrotic Metal Detector

We utilise MTF-1 as a key component for the innovative cellular metal sensor. MTF-1 (metal-responsive transcription factor-1) is an important transcription factor that plays a crucial role in the recognition and cellular response to heavy metals as well as in the maintenance of normal metal homeostasis.[41, 42] In response to heavy metals such as cadmium, zinc and copper, MTF-1 induces the expression of metallothioneins and other genes involved in metal homeostasis.[43]

Signaling pathway of the heavy metal biosensor

MTF-1 is known as a nucleocytoplasmic shuttle protein, which under normal conditions is present in both the nucleus and the cytoplasm, but accumulates in the nucleus upon accumulation of heavy metals. There it binds to promoters that contain a metal-reactive element (MRE) and thus specifically regulates gene expression.[43, 44] This regulatory ability allows MTF-1 to control cellular adaptation to various stress conditions, especially during exposure to heavy metals, but also during hypoxia or oxidative stress.[41]

For our project, we have introduced MTF-1 into HEK cells together with a designed highly active promoter with a specific fluorescent target gene whose expression is induced by the activation of MTF-1. Through this method, we were able to generate a fluorescent signal that allows us to accurately measure the presence and concentration of metal ions in the environment.

Further Investigation by Spectroscopic Measurements and Computer Models

In addition to the cellular approach, controlled studies are employed to assess the functionality and complexation of metals with antibiotics. Through spectroscopic investigations via Raman and Fourier Transform Infrared Spectroscopy as well as our own development of a sensor using light-based detection via whispering gallery modes, we are complementing all measurements. To find out more about this, click here.

Furthermore, computer models are utilized to predict how various factors like antibiotic residue concentration and antibiotic interaction influence the chromosomal mutation and horizontal gene transfer of bacteria or how antibiotic resistances are influenced by the presence of metals in the water. We simulate the dissemination of antibiotics and heavy metals in liquid samples and illustrate their impact on the sensor system in detail. To have a look at our models, click here.

Why We Have Chosen This Project

Our project achieves early detection of heavy metals and antibiotic residues in liquid samples and improves the understanding of the biological interactions between our sensors and substances, thereby promoting technological advancement and ethical responsibility towards environmental and human health protection. The integration of clever genetic engineering, such as orthologous expression of PknB or MTF-1 based metal detection, will facilitate the detection of heavy metals and antibiotics.

Our previous research during our studies, as well as our Bachelor's or Master's theses in the context of implant research at the NIFE (Lower Saxony Center for Biomedical Engineering, Implant Research and Development) or human medicine at the MHH (Hannover Medical School) have revealed the challenge of resistances in the clinical environment. We quickly recognized the critical need to address the problem of water contamination. Our research for a project focused immediately on antibiotics and metals, inspired for example by previous work of the iGEM team from Frankfurt 2023. We were driven to improve the detection of these contaminants in liquid samples using synthetic biology.

Synthetic biology is the ideal solution to realize our goal of a single cellular approach for both metal and antibiotic detection, as it allows us to develop an orthologous expression of PknB and MTF-1 in one biosensor system. This enables flexible and wide-ranging application in the respective fields of operation. 

During the engineering cycle, we aim to revise our sensor concept several times with the help of our experiments and measurements to achieve the best possible result for our cellular sensor. During the different phases of testing and learning, both the computer modeling and the dry lab results will help us to validate the sensor, as well as to improve the cellular design.

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