The use of pharmaceuticals has steadily increased over the years, and is expected to rise further due to the rise of life expectancy and growing global population. Once excreted, the resulting pharmaceutical waste products end up in sewage systems and surface water. As a consequence, the concentration and the range of pharmaceutical waste products in our surface water has steadily increased. Even our drinking water contains these pharmaceutical residues, since drinking water is obtained from surface water in many countries, including the Netherlands. This has already posed problems to water-living animals, like fish, and will affect us as well, if left untreated. A prerequisite for drinking water purification is a sensitive detection system capable of rapidly detecting a broad range of pharmaceuticals in water.
Current methods to detect and measure pharmaceutical waste levels in water utilize chemical analysis methods such as mass spectrometry, or bioassays. Chemical analyses are the current method of choice, but they are usually expensive and time consuming. Bioassays are more affordable and can be easier to implement, but their development is still in its infancy. Here, we have developed DeTaXion, an accurate, fast, and affordable bioassay to detect pharmaceutical waste products in water samples. The low cost of DeTaXion will facilitate frequent sampling, increasing the ability of water treatment facilities to accurately and rapidly respond to changing contaminant types and levels.
Applications of DeTaXion
The low cost and rapid assay time of DeTaXion makes it a versatile platform, that can be used as a stand-alone product to continuously monitor a water supply, or to complement traditional chemical analyses
DeTaXion to ease and cheapen chemical analysis
Chemical analysis using mass spectrometry is routinely used to determine the presence and concentration of medical waste products in water. This expensive analysis is often used on samples that turn out not to be contaminated. Therefore, a rapid pre-screening method to select for only those samples with contaminations would drastically reduce costs. DeTaXion can play a crucial role here, rapidly selecting samples contaminated with a particular class of chemicals for further detailed analysis (Onno Epema, Rijkswaterstaat; Marlies Kampschreur and Maarten Nederlof, Waterschap Aa en Maas).
Furthermore, DeTaXion can also ease the interpretation of chemical analysis data. Since the biosensor will only measure the presence of specific groups of compounds, such as antidepressants and related hormones, it provides an indication which compounds should be searched for during further chemical analysis. In other words, only a limited set of chemical contaminants needs to be further investigated in a selected number of samples, in contrast to an expensive and time consuming broad screen.
DeTaXion facilitates performing a larger amount of measurements
DeTaXion is low-cost, convenient to use, and enables local sampling. On site measurements enable a broad range of local applications, from Dutch water authorities to medical caretakers and communities wanting to monitor their water supply.
For water authorities, the on site detection abilities of DeTaXion enables confirmation of pharmaceutical waste concentration predictions in specific areas, and the confirmation of national measurements. In this case a set of one or more medicine residues can be used as a standard to indicate local concentrations (Marlies Kampschreur and Maarten Nederlof, Waterschap Aa en Maas). Furthermore, DeTaXion can also be applied in water treatment plants to continuously measure the efficiency of water purification (Marijan Uytewaal-Aarts and Marlies Verhoeven, HRSR).
Local sampling can also provide information on the health of the the water supply in a specific neighborhood, for example measuring the concentration of the commonly found pharmaceutical waste products oestrogens and diclofenac (Marlies Kampschreur and Maarten Nederlof, Waterschap Aa en Maas). Finally, DeTaXion can be used by medical caretakers to determine the efficiency of medicine uptake of patients by measuring drug residue levels in urine (Marlies Kampschreur and Maarten Nederlof, Waterschap Aa en Maas).
In order for DeTaXion to be a safe and convenient device, there are several problems that have to be overcome. These problems can be divided into three categories: regulatory restrictions, technical problems and safety issues.
There are several regulations designed by the European Union on the production of devices containing GMOs, which were followed strictly. This resulted in our decision to limit the use of our sensor to closed environments preventing unwanted breakouts and introduction into the environment.
Based on interactions with stakeholders and experts , we composed a set of requirements to our device to aid accurate, convenient and fast measurements.
|Representative samples||In order to obtain a representative sample, a certified method, such as the NEN-6600-1 method should be used. (Roberd Boer, Tauw).|
|Water filtering||Samples have to be filtered so there are less large-size contaminations interfering with the measurements (Peter Behnisch, Biodetectionsystems; Martin van der Berg Utrecht University; Marijan Uytewaal-Aarts and Marlies Verhoeven, HRSR). Therefore a filtering step is included in the device.|
|Multiple compounds||Stakeholders emphasized the need for a detector that can measure groups of compounds. This will be achieved by offering a customizable set of E. coli strains incorporated in the design. In case detection of a very high number of compound variants is desired, measurements can be done in the lab (Onno Epema, Rijkswaterstaat; Roberd Boer, Tauw).|
|E. coli survival||We are using the K12 lab strain in our experiments, which is a quite delicate strain, to limit environmental risks. Therefore temperature and pH fluctuations have to be taken into account. It is also imperative to characterize the effects of the plasmid used in our bacteria, to determine the effects the plasmid could have on the survival of our strain. (Gert-Jan Euverink, Wetsus; Marijan Uytewaal-Aarts and Marlies Verhoeven, HRSR)|
|Plasmid preservation||It is important for the accuracy of the system, that the bacteria retain their function and therefore their plasmid. In order to assure the plasmid preservation, the device will contain an antibiotic, while the plasmid will express an antibiotic resistance gene (Gert-Jan Euverink, Wetsus). A future version of the device may use an auxotrophic marker instead, to reduce antibiotic use.|
|Rapid measurement||After the addition of a sample to the E. coli , BRET measurements need to be done within seconds, since the response of the pathway is very rapid. This is incorporated in the design, by measuring luminescence during sample addition.|
|Handheld-device||To facilitate quick, on-site measurements, the filtering and measuring methods should not be a limiting factor.|
|Toxicity of the samples||In order to make sure the bacteria have not died instead of a pathway inactivation, a positive control will be used in the form of an E. coli strain constantly expressing luciferase (Martin van den Berg, Utrecht University).|
|Specificity||Limiting the interference of other compounds with the measurements is essential. Therefore, it is important to determine the specificity of the receptor for specific compounds in advance.|
|Clear data||To ease the usability of the data, a software program will be designed that directly transforms the data into easily accessible compound concentrations (Jos van der Vossen, TNO).|
It is crucial that the used E. coli are disposed in a responsible, safe manner (Gert-Jan Euverink, Wetsus). To accomplish this, agents such as bleach will be added to ensure the complete destruction of E. coli , such that the device can safely be disposed of in accordance with local regulations.
Another consideration critical for the safety of the device is the prevention of bacterial escape (Martin van den Berg, Utrecht University). To mitigate this danger, we are using a laboratory strain (K12), which has minimal chances of survival in the environment. An additional measure will be taken in the form of a kill switch. The E. coli will be modified to produce toxins, while the device contains a compound inducing the expression of anti-toxins. In case of an escape, the (very unlikely) survival of our E. coli can easily be detected by PCR amplification of sequences unique to the DeTaXion strain (Marijan Uytewaal-Aarts and Marlies Verhoeven, HRSR)
Taking all considerations into account, we designed DeTaXion in such a way that we are confident that it assures all our considerations. This resulted in the following device:
The DeTaXion device consists of three main components: (1) a multi-pipette component with a kill switch to introduce the sample; (2) a sealed, light-proof 96-well plate containing the DeTaXion core: the modified bacteria; and (3) a sensor to measure the bioluminescence signal. The use of 96-well plates allows for a high level of customization to different applications. Modified DeTaXion bacteria can be included that respond to different compounds, or that are sensitive in a different concentration range. Furthermore, not all 96 wells have to be measured at once, as multi-pipette components can be ordered for one or more rows in the plate.
The multi-pipette component is introduced to the plate, piercing the seal. Rubber gaskets allow the plate to be sealed off completely, keeping the modified DeTaXion bacteria securely contained.
A potentially contaminated water sample is introduced into the main reservoir of the pipetting device. The sample is filtered to remove larger compounds that may impede accurate measurement.
The sample is evenly divided over the wells containing DeTaXion bacteria.
Next, the sample can be introduced into the 96-well plate by pressing the eject button on the handle
When the wells are loaded, the bioluminescence measurements are performed and the results are displayed on an application also available for smartphones connected to the measuring device.
Once the measurements are completed, bleach is added by pressing the kill switch. This will kill the bacteria, preparing for safe disposal.
Finally, the device can be safely disposed of in accordance with local regulations. Where appropriate, the multi-pipette component can be returned for re-use.
The apocalypse has begun