SAFETY

To ensure the safe development and implementation of our biosensor, SPYKE, various risk assessments were performed in and outside the laboratory, as well as an analysis of the intended implementation site. Here, we present these risk assessments, among others the Safe-by-Design strategy, that were used to identify and analyze risks associated with our product SPYKE.

Introduction

Safety is an essential part when developing a product like ours. Safety doesn’t only include anticipating and mitigating risks pertaining to health and/or the environment, but should also take into account ethical, social, and emotional aspects. To consider all aspects related to safety, we opted for a Safe-by-Design (SbD) approach, a way of managing risk by integrating design choices specifically for safety [1] . We talked to a variety of stakeholders about the safety aspects of our project, particularly the Dutch National Institute for Public Health and the Environment (in Dutch: RIVM ) who helped us analyze our project and integrate an SbD approach. An overview of all the components and the mechanism of SPYKE is shown in Figure 1. When GHB enters the glass it diffuses through a filter to a department with DNA-bound BlcR proteins. GHB binds to BlcR subsequently unbinding them from DNA resulting in a capacitance change that turns on a warning light. For a more extensive look into the mechanism, the components of SPYKE, or the stakeholder interactions see the project description , hardware , and the integrated human practices pages respectively.

An overview of all the components and the mechanism of SPYKE.
Figure 1. An overview of all the components and the mechanism of SPYKE.

Safe-by-Design

We used the SbD approach to identify various potential risks and possible ways to lower or mitigate these. Risks were identified using desk research and in the case of insufficient knowledge by means of interviews [1] . We talked to various stakeholders such as food safety institutions and the Dutch National Institute for Public Health and the Environment (RIVM). Mitigation strategies were developed for the identified risks consisting of design changes, adaptations in the proposed implementation, and proposed research to improve safety. Figure 2 shows an overview of the risks and mitigation strategies considered in our safe-by-design.


The potential risks and mitigation strategies resulting from the safe-by-design approach.
Figure 2. The potential risks and mitigation strategies resulting from the safe-by-design approach.

Food safety risks

The most important risk that should be minimized for SPYKE is related to food safety. All the components in contact with the beverage should be food-safe, which means there are no microorganisms, chemicals, or materials present in quantities that can be harmful to human health [2] . To accomplish this we made design choices and changes to make sure all the compounds in contact with the beverage are considered food safe, and the unsafe compounds cannot come into contact. We integrated the feedback from experts from five different food safety institutions into our design. Check all the interviews in the integrated human practices page.

  • European Food Safety Authority (EFSA)
  • Wageningen Food Safety Research (WFSR)
  • Ministry of Health, Welfare and Sport (VWS)
  • Netherlands Food and Consumer Product Safety Authority (NVWA)
  • Bureau of genetically modified organisms (Bureau GGO)

Filter

The filter present in the glass is in continuous contact with the beverage. We considered using ultrafiltration discs or dialysis membranes for this. However, ultrafiltration discs are meant to be used under pressure for a short period of time [3] as opposed to dialysis membranes which are meant to be used without pressure for a long time [4] . To ensure the filter works safely throughout the night we opted for using dialysis membranes. Hidde Rang informed us that the dialysis membrane falls under the category of food contact materials, which are subject to different rules [5] . We chose a dialysis membrane made from regenerated cellulose as it is considered safe as a food contact material [6] . The regenerated cellulose also adheres to the guidelines provided by the European Union [7] .

BlcR

The BlcR protein must come into contact with the beverage without contaminating it for SPYKE to perform safely. Since the protein is partially unbound and only partially retained by the cellulose filter, it has the highest likelihood of ending up in the liquid. According to the Wageningen Food Safety Research (WFSR ), proteins may be toxic or allergic. We carried out bioinformatics screenings for toxicity and allergenicity and made design choices to reduce the possibility of BlcR ending up in the beverage to ensure that the use of the BlcR protein does not compromise the food safety of SPYKE. The outcome of the bioinformatics screening was that the BlcR protein showed no indication of toxicity and allergenicity.


To be sure BlcR doesn’t exhibit toxicity or allergenicity the WFSR recommended doing bioinformatics-based toxicity and allergenicity screening. Using the amino acid sequence and the structure of a protein an indication of the risks of adhering to toxicity or allergenicity can be given.

Toxicity

A prediction of hazards associated with proteins can be done by comparing our protein of interest to proteins with known associated risks. The WFSR told us to take these steps to screen for potential toxicity of our protein:

  1. Download the Fasta file or sequence of your protein.
  2. Download the Fasta files of proteins associated with the term toxin on UniProt which are checked by SwissProt. https://www.uniprot.org/uniprotkb?facets=reviewed%3Atrue&query=toxin.
  3. Blast your protein against the downloaded Fasta files. You can use an online BLAST software like viroblast. https://indra.mullins.microbiol.washington.edu/viroblast/viroblast.php.
  4. For all the identified proteins with a similar sequence, check through their description on UniProt if they are actually toxins and not toxin-associated proteins.
  5. From the remaining proteins check the sequence overlap. Discard the protein if there is no large sequence overlap or very similar structures. The proteins with a large sequence overlap can indicate a risk of toxicity.
  6. For proteins with similar structures research the protein if these structures are involved with the toxicity. If this is the case there is also an indication of a risk of toxicity.

Allergenicity

A similar approach can be applied to check for potential allergenicity. The WFSR recommended two websites for this. You can use http://www.allermatch.org to check for similarities between common allergenic proteins and http://www.allergenonline.org/celiachome.shtml to check for celiac-associated proteins.



Small volumes

We tried to minimize the protein concentration inside the beverage by lowering the volume of the BlcR solution as much as possible. A lower volume of BlcR solution compared to the beverage volume decreases the amount of protein that can diffuse into the beverage. We adapted our prototype to a three-layer system with a designated space for the electrodes and BlcR solution. This way, the least amount of BlcR solution is needed.

Colored or bitter compound

Hidde Rang advised us as an extra safety measure to include a bitter or colored substance inside the protein solution as a warning signal if the filter malfunctions. We chose to include a colored substance instead of a bitter one for two reasons:

  • Very strong bitter molecules are often toxic and tasting it can be an unpleasant experience [8].
  • Bitterness can be hard to detect for intoxicated people, especially when mixed with sweet cold drinks. Bitterness can also only be sensed by the consumer of the drink while a color change can also be observed by bystanders.
We propose to use small colored food-safe beads as a warning signal, but the best optimal system should be investigated [9].

Oligonucleotides

Other molecules that have a chance of contaminating the beverage are the oligonucleotides. The probability of this happening is relatively small as the DNA is surface-bound using thiolation and has a molecular weight higher than the molecular weight cutoff of the filter. Research shows that thiol-modified DNA has relatively high stability over a couple of days, meaning that ethanol, low temperatures, and low pH found in beverages don’t decrease the stability [10]. DNA is also considered food safe [11]. Even though our DNA is considered a GMO, research suggests GMO-DNA is also food safe. We still asked the WFSR about the food safety of DNA and they told us this DNA would not be able to propagate in the human body as it is degraded.

Plastic

We initially thought of using a 3D printer to create the cups for SPYKE just like with our prototype. This would result in a food safety risk as 3D-printed materials can be toxic when used over a longer period [12]. Particles from the materials can migrate into the food, and the shape of 3D-printed materials allows for bacterial buildup. Together with a decrease in cost, this was a reason we opted for mass-producing the SPYKE cups from food-safe plastic recycled PET [13].

Electronics

The consumption of electrical components inside our sensor should be avoided as they are toxic. To avoid any chance of this happening, there are three protection layers present in the glass. The first is the filter meant to prevent proteins from diffusing inside the beverage. The second is a watertight seal between the protein solution and the electrical components. The last protection layer is an extra seal around the battery as an extra protective layer around the toxic battery [14].

Mercaptohexanol

Initially, we wanted to use mercaptohexanol as a coating agent for the gold electrode to prevent unwanted binding. The WFSR told us that mercaptohexanol is an irritant and causes a bad smell. Therefore, in our final product, we propose to use very short DNA oligonucleotides to cover the surface without blocking the binding of BlcR. Further experiments should be done to test if this is indeed an adequate replacement.

Waste risks

The use of SPYKE can result in waste which can cause biocontamination and environmental damage. The biocontamination would consist of the ampicillin resistance plasmid used to produce BlcR contaminating the environment. The environmental damage would be caused by the one-night components of SPYKE.

Gain-of-function by ampicillin resistance genes on plasmid

As identified by the dual-use scan, the E.coli strain with an ampicillin resistance gene can result in a biosecurity risk. The E.coli contains a plasmid with an ampicillin resistance gene, necessary for amplification during biosensor manufacturing. The ampicillin resistance gene could potentially give an evolutionary advantage, therefore resulting in plasmid uptake by microorganisms when the biosensor is utilized outside the ML-1 designated laboratory spaces. Hence, the microorganisms could get gain-of-function such as resistance to antibiotic medication.

To minimize the risk of spreading the ampicillin resistance plasmid the BlcR protein is purified before leaving the production facility, resulting in no plasmids leaving the factory. Cecile van der Vlught also explained this would not be a large problem as ampicillin is not a last-resort antibiotic and is used substantially in research.

Environmental risks:

Using SPYKE will result in waste as certain parts cannot be reused after usage. This will have negative consequences on the environment. The production of waste when using SPYKE should be minimized. The design and proposed usage of SPYKE were altered to achieve the goal of minimal environmental risks.

Design changes

The design of SPYKE was altered to minimize the number of components that can be used for only one-night while still being able to function. We created a three-layer cup that can be unscrewed into three separate parts. The electronics in the bottom layer can be easily reused, and the one-night-use components can be replaced to recycle the plastic. For a full description and 3D model of the three-layered cup see the hardware page. The only remaining one-night use components used in SPYKE are the DNA electrodes, protein, and filter as they can lose their functionality after one night. The filter will dry out after it has been used [15], the protein will degrade [16], and the DNA will dissociate [17]. These components can in all likelihood be used for longer than a night which will reduce the waste, but to ensure a well-functioning system initially they will be recycled after a night.

Proposed usage

To make sure the glasses are re-used as much as possible we propose a recycling system to return the glasses. After the user finishes the drink to get a new glass the old glass should be returned to make. This procedure has been used for some time to lessen plastic waste, not only in the Netherlands but also in other nations, particularly at festivals [18]. For more information read the proposed implementation.

Usage risks

The design of our tests, particularly the chance of false positives and negatives, can also be a cause for risks. Also, the risk of missing the output signal should be taken into account. The sensor should also be handled well by users and the nightlife establishments. Lastly, the security of the data from the sensor should not be compromised.

False positives and negatives

False positives and negatives would have a great impact on the user of SPYKE in a variety of ways. As mentioned by stakeholders and literature, especially ethanol can be a cause of false positives in GHB tests [19] [20]. False positives can induce unnecessary fear or medical treatment [20] for users and can cause false accusations of people or venues. False negatives can leave people unprotected and prevent them from getting justice [21]. To minimize the risk of false outcomes of our test, we decided to use an electrical output system. With an electrical system, it is possible to set a threshold for the minimum amount of change in signal to trigger an output. This is important as the endogenous presence of GHB in some drinks [22] and in one’s body [23] could result in a positive output through the leakage. With a biological system, the risk would be higher as it can be hard to distinguish between a short reaction with a high concentration and a long reaction with a low concentration.

To minimize the risk of false positives we used a system that measures the difference between two almost identical capacitors. The only difference between them is that only one of the capacitors is responsive to GHB. As explained by Jeroen Bastermeijer this limits the influence of environmental perturbations on our system thereby lowering the chance of false positive and negative outputs [24].

In terms of design choices, we also opted for the glass over one’s tooth for the sensor’s placement to minimize the consequence of a false positive. When a false positive output occurs on the tooth, the user has already consumed the drink. This will cause a lot of unnecessary stress on the user and the people around them. When the sensor is in the glass, these troubles can be partly avoided, because the false positive output will be given before the drink is consumed.

Missing the signal

There is the risk of a user missing the signal. This could result in a case of spiking which could have been prevented. To minimize the chance of this happening we adjusted the sensor placement and output signal.

Sensor placement

Our first design choice of having the sensor placed on your tooth. By hosting a focus group (N=10) and handing out a survey (N=661), we found that people preferred to have the sensor in a glass. One of the most frequent reasons was that the glass was safer, as the sensor on your tooth would warn you after the drink was (partly) consumed. By placing the sensor in the drinking glass, this problem can be avoided as the positive output signal can be given before the drink is ingested.

Output signal

We first opted to have a Bluetooth output signal to the phone of the user, the club, and an emergency contact. Eerste Hulp Bij Drank en Drugsongevallen (EHBDD) (First Aid for Alcohol and Drug Accidents), clubs, and the police recommended using a LED as an output. The reason for this was that the Bluetooth signal could be easier to miss for the user and the clubs. Also, it can be hard for the club to find the glass with the positive signal. To avoid these troubles, a LED will be used as a positive output signal as it will be immediately seen by users and the nightclub.

Data security

The data the sensor collects should be private as public information on victims of sexual assault can lead to discrimination against the victim. They can be shamed, accused of lying, and even denied jobs or housing [25]. Our initial idea was to have a Bluetooth output that was sent to your phone, an emergency contact, and the nightlife establishment. This brought the risk of the Bluetooth signal being hacked [26]. This was one of the reasons we switched to a light output as no data is collected in the device and no signal is sent to phones. The data will now be collected by the police when they get notified a glass gave a signal and stored on their secure network.

Non-proper handling

To function as intended SPYKE should be handled well by the user and the nightlife establishments.

  • The user should know what output is to be expected when using SPYKE, how to recycle the system and safety precautions that should be taken when using SPYKE.
  • The nightlife establishment should know how to clean SPYKE, how to act when the sensor goes off, and how to explain what SPYKE is.
To give quick access to this information, Lars van Driel suggested including a QR code on the glasses. This QR code can be scanned and a user manual will be provided.

Recycling

Initially, we thought of sending the required components to recycle SPYKE to the nightlife establishments. This would greatly reduce the cost and emissions of SPYKE as the sensors didn’t have to be shipped back and forth. Human error contributes to a large number of errors in electronics production, especially in high-stress environments like nightlife [27]. To make SPYKE as reliable as possible, we opted for recycling the SPYKE cups inside the factory by specialists to reduce human error and include validation tests.

SPYKE for individuals

As heard from the victim without proof and more stories, they suspected the bartender of spiking their drink [28] [29]. As suggested by the victim and Aaldrik Krol we opted for also selling SPYKE to individuals and offering a service to return and recycle it.

Societal risks

The existence and application of our test should not impose any harm on society. For instance, it should not steer the perpetrators to using other drugs that cannot be traced by means of our test, other ways of administration such as needle spiking, or specifically targeting people that do not appear to be testing for drugs.

Other drugs

The existence of a GHB test can cause a shift in the preferred drug used for spiking. The Dutch Forensic Institute mentioned the existence of other drugs which can be used for spiking. An example was synthetic benzodiazepines, which can be very strong [30]. Future research should be done to test whether biosensors for other possible rape drugs could be implemented with our GHB sensor. Electrochemical detection of benzodiazepines [31] and ketamine [32], two other common rape drugs [33], has already been proven possible. These detection mechanisms could possibly be combined with our electrical sensor. Due to time constraints, we could not test this, but this possibility should be investigated. A paper from 2019 [34] suggested the possibility of using directed evolution to create novel allosteric transcription factors. Further research could be done to create allosteric transcription factors for possible rape drugs. These transcription factors could possibly be added to our sensor to screen for more drugs.

Needle spiking

Another risk is that perpetrators use another way of administering the drugs than spiking one’s drink. The phenomenon of needle spiking is on the rise, as largely reported by the media [35], and it consists of injecting the drug through a small needle. However, as for normal spiking, there is little proof. We talked to the police, the Trimbos Institute, and more experts. They doubted that needle spiking would become a large problem, for more information see IHP. Till now there is almost no proof of needle spiking happening on a large scale, but we could use our mechanism to test for the occurrence of needle spiking. This could be done by using our mechanism in a quick test for GHB in possible spiked people for more information see the proposed implementation page.

Target people without the test

The police warned us that there was a risk that the people who weren’t using our sensor could be targeted. If the sensor is easily recognized, perpetrators can take advantage of this by spiking people that are not using the sensor. On the other hand, we heard from a victim they were presumably spiked by a drink someone bought for them. They suggested having some way of recognizing the glass as an SPYKE glass. To find a compromise between the advice of the police and the victim we attach the QR code with the user manual at the bottom of the glass. This way it’s easy to recognize the glass for the user and hard for other people.

Biosecurity

Biosecurity is one of the key facets of the iGEM competition and should be carefully considered when working on a project [36]. Dual-use procedures and practices can be a good way of identifying biosecurity risks [37]. Possibilities of misuse of biological research and product development can be easily overlooked as it can be hard to escape one’s own worldview. The Dutch National Institute for Public Health and the Environment developed a dual-use quick scan consisting of a 15-question inquiry. The questions cover possible dual-use characteristics and can be answered with: “Yes”, “No” or “Unknown”. Questions answered Yes” or “Unknown” can indicate a possible dual-use characteristic of your research [38].

We conducted this quick scan twice together with Cecile van der Vlught. Once for the implementation and once for the production of SPYKE. Concluded from the first scan there are no dual-use characteristics identified for the use of SPYKE. As we use a purified protein and short DNA oligos which the WFSR said could not propagate in nature. As shown by iGEM TU Delft 2021 the use of a biosensor could result in a dual-use character by misuse of personal data. As further explained in the safe-by-design approach data security will not play a role as our device doesn’t collect any data, but the police will.

The second dual-use scan regarding the production of SPYKE resulted in one “Yes” to the question: “Is it likely that your research will increase the resistance of the biological agent to clinical and/or agricultural prophylactic or therapeutic interventions, including antimicrobial resistance?”. This is because of the use of an E.coli strain with an ampicillin resistance gene to produce BlcR. Cecile mentioned this would not result in a biosecurity concern. This is further elucidated in the biosecurity section of the safe-by-design.

Lab safety

General safety

Our laboratory is located in the Biotechnology department of the Applied Science faculty at TUDelft, see Figure 3 and 4. The laboratory is in full compliance with the ML-1 lab requirements and the iGEM safety and security policies. We designed all our experiments to be done at the ML-1 biosafety level and solely utilized microorganisms classified in this risk group. To reassure that our team members have the required knowledge for emergency operation procedures and to perform the laboratory work safely, all of our team members passed the following safety tests:

  • General building safety (meeting points, emergency numbers, flight plans, etc.).
  • General laboratory safety (chemicals, waste disposal, clothing, safety precautions, etc.).
  • Biological safety (ML-1 grade safety, biological waste, safety precautions, etc.).
  • Laser safety (general safety precautions).

Next to that, we also followed lab training sessions given by our supervisors. Within this lab training, we also learned how to discard different types of waste in the correct disposal bins, how to handle dangerous chemicals and how to safely perform certain standard lab experiments. Before being allowed to perform any experiments in the lab, a general safety report covering all the involved activities, chemicals, organisms, and other relevant safety information was handed in and received approval by the Institution's Biosafety Officer. All the acquired biological parts (genes, plasmids, strains) were first registered in the Institution's local repository and received approval before being ordered.

TU Delft laboratory.
Figure 3. TU Delft laboratory.

TU Delft laboratory.
Figure 4. TU Delft laboratory.

Biosafety

Strains

For our project, E.coli DH5𝞪 are used to store the BlcR plasmid and E.coli BL21(DE3) will be used to express the plasmid containing BlcR. These E.coli strains can be utilized at the ML-1 biosafety level.

Cell-free system

A cell-free system (PUREfrex 2.0) was also used to express the BlcR mutants. This is a safe-to-use system. This system is not classified to GHS classification. By following the ML-1 lab protocols and regulations, we avoided harm to laboratory staff.

Vectors and inserts

To produce BlcR, the BlcR sequence was expressed in BL21(DE3) cells by transforming them with a pET-11a plasmid containing the BlcR gene as an insert. The expression will be induced using isopropylthio-β-galactoside (IPTG). These plasmids also contained an Ampicillin resistance gene, which made the selection with ampicillin possible. All features of these plasmids are in compliance with the ML-1 regulations.

Chemicals

Some hazardous chemicals were used, such as SYBR Safe. The incorrect handling of carcinogenic and toxic materials could result in exposure and negative health impact on the users and third parties. Additionally, incorrect disposal could result in a risk for society and the environment. Therefore, all of the material handlings are to be performed with adequate gloves and visual aid on the correct disposal flow available in the area for correct waste management.

Controlled substances

Our project revolved around the rape drug gamma-hydroxybutyric acid (GHB). However, GHB is described as a List 1 molecule under the Opium Law in the Netherlands. Therefore, no person or institute can work with GHB without a permit for these molecules. Unfortunately, TU Delft is not in possession of this permit. Because of this, an analog, based on molecular similarity has been searched for. Based on the chemical similarity between succinic semialdehyde (SSA) and GHB and the previous knowledge that BlcR is also SSA, SSA has been chosen as a legal analog to experiment with [39]. The structure of SSA can be found in Figure 5 . In the final stage of our project, we conducted validation experiments using our final prototype and GHB. These tests were conducted at the Trimbos Institute under supervision, which is permitted to work with GHB.

Molecular structure of succinic semialdehyde (SSA).
Figure 5. Molecular structure of succinic semialdehyde (SSA).

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