“Exploring the cell”: Α Βiology-related Αrt & Craft Workshop for Children in the Natural History Museum of Crete

The Greek educational system does not provide enough material for biology concepts at early education levels, especially in primary school. Considering our mission to talk about synthetic biology and biology in general, we decided to address children to enrich their knowledge of basic biology concepts and see life from a different perspective, a microscopic one. Thus, we taught them about the smallest unit of life, cells. In collaboration with the Natural History Museum of Crete, we organized an interactive workshop in which we applied an educational approach based on storytelling, playing, and drawing. The event aimed to give children a taste of basic biological concepts and allow them to learn about the organelles of the cell and their functions in a playful way.

At first, we gave our little friends a presentation about animal and plant cells. After this lesson, it was time for action. We separated in small groups and used a stereoscope to see cells up close. This hands-on experience helped children to understand the size scale of cells. To determine whether or not the children understand the differences between plant and animal cells, we provided them with handmade puzzles of human and plant cells to solve. Then, we encouraged every kid to draw their imaginary cell using their creativity and personal style. This activity helped them assimilate their new knowledge and remember that day by taking their unique drawing home. The feedback from the children and their parents, who filled out an evaluation form, showed us that not only did the children have a great time, but we also managed to strengthen their thirst for learning about biology.

Meet iGEM uniCRETE: An Informative Event for Students of University of Crete

The School of Sciences and Technology of our University provides a thriving environment for the growth of inquiry and invention. Our fellow students are always interested in learning about evolution in the world of science. Since synthetic biology is a relatively new concept, we realized that only a few students are familiar with it. That is why we organized a meet-the-team event in our university’s facilities to inform students about synthetic biology, the iGEM Competition, and our project in detail. We approached the definition of synthetic biology entertainingly by creating a Kahoot game. Then, we introduced the iGEM competition to the audience through informative presentation slides and explained how our team collaborates and works, by analyzing the duties of each subteam in detail. The attendants had the chance to ask specific questions about our project and join an open conversation about the overuse of pesticides in the agriculture industry.

Leveraging Social Media for Synthetic Biology Education

In today's interconnected world, the influence of social media cannot be overstated. It has fundamentally transformed the way we connect, share knowledge, and collaborate. Within our team, we recognize the profound impact platforms like Instagram, Facebook, LinkedIn, and TikTok have on our mission to bridge the knowledge gap in Synthetic Biology education. Our journey into the world of social media has been extraordinary, weaving together threads of engagement, education, and outreach to enhance the human experience and further our mission. Even though it was the first year that iGEM uniCRETE became part of iGEM’s family, we were eager to share every milestone, news update, and event with the world, reflecting the collaborative and communicative spirit that defines an iGEM experience. We have shared a diverse range of content, from revealing what a day in the life of an iGEMer seems like to delving into the details of our innovative project, "DeltaSense". These efforts have opened a door for the public to explore the world of iGEM, letting our scientific enthusiasm reach beyond the lab’s walls

Collaborations with fellow iGEM teams on various educational subjects, from projects related to World Biodiversity Day to creative videos offering a glimpse into a day in the life of an iGEMer, have brought us closer to a worldwide scientific community. Engaging biology-themed quizzes on topics like health, wildlife, DNA, and pesticides have connected us with our audience while surveys on ethical matters surrounding inclusivity reflect our dedication to building a diverse and welcoming scientific community. Leveraging technology, we've transcended geographical boundaries, establishing global connections and partnerships.

In terms of aesthetics, we've embraced vibrant and playful elements in our social media presence. Captivating hand-drawn posts, lively colors, and a modern visual style aim to challenge the misconception of science as dry and inaccessible. Instead, we strive to convey that science is a source of wonder and delight for all. Through carefully curated content and aesthetics, we aspire to inspire a new generation of thinkers and innovators.

Fostering Global Connections for Synthetic Biology Education

As an integral part of the iGEM competition, we understand the importance of creating global connections and participating in the worldwide scientific knowledge exchange. Even though the medal criteria have changed this year and the collaborative events with the rest of the iGEM team are not prerequisite, we still wanted to join forces with other iGEM members in order to broadcast science all over the world. Through our proactive and dynamic approach, we are confident enough to claim that we both expanded our project and enriched the iGEM community by means of international teamwork.

Collaborations by iGEM uniCRETE:

This year, our team fostered two collaborations, both aimed at advancing synthetic biology education and outreach.

World Biodiversity Day Tribute

On the 22nd of May, teams: Bulgaria, Freiburg,NU Kazakhstan, Thessaloniki, Neotech-e, Patras Biology, ABOA, IONIS, IISERP, PTSH-Taiwan (ButeurACE), NTHU-Taiwan, Thrace and Scotland joined us in celebrating World Biodiversity Day. The reason we chose to initiate this collaboration was our desire to raise awareness about special local species and convey our determination to support the protection of wildlife. After all, we expected our digital footprint to correspond to the principles that inspired us into creating DeltaSense, which targets a substance known to threaten biodiversity. Therefore, our instagram feed gladly became a welcome habitat for numerous extraordinary creatures of this world.

TikTok Collaboration - "A Day in the Life of an iGEMer"

On the social media platform TikTok, we collaborated with four other iGEM teams to create a one-minute video titled "A Day in the Life of an iGEMer." These creative collaborations merged the latest internet trends with our passion for biology, providing insights into our iGEM-spirited lives. The objective was to motivate individuals interested in future iGEM competitions or pursuing careers in science, in a fun and relatable way.

Collaborations iGEM uniCRETE participated in

Recognizing the immense value of collaboration and scientific exchange with other iGEM teams worldwide, we were fortunate to participate in several meaningful collaborations initiated by other teams this year. These collaborations exemplify our dedication to working together and our mutual drive to push the boundaries of science. Even though the concepts are not closely related to our project, the collaborations proved themselves as just another way to promote iGEM Competition’s mission and problems solved by Synthetic Biology all over the word. Here's a breakdown of our experience in the field:

• Patras Medicine iGEM 2023 World Health Day Collaboration In this collaborative effort, iGEM teams from across the globe united to convey a powerful message, advocating for universal access to healthcare and well-being on World Health Day. It was a global initiative to ensure that everyone, regardless of their location, can achieve the highest level of health and well-being.

  

• iGEM Thrace 2023 “iGEM is …” Video We teamed up to create a collaborative video that not only found its place on social media but was also displayed at the first Panhellenic Biosciences Student Conference held in Alexandroupoli. This video beautifully encapsulated what iGEM represents to us, providing a special view on our collective experience.

• iGEM Bulgaria 2023 Icebox Collaboration Each participating team had the opportunity to decorate their own icebox and craft a short story related to it. This collaboration added a creative and artistic touch to our scientific endeavors, allowing us to express our perspectives in a visually compelling way.

  

• iGEM SVCE Chennai 2023 World Nature Conservation Day Collaboration On World Nature Conservation Day, iGEM teams from around the world came together for a noble cause. We collectively planted a tree as a symbolic gesture of our common goal to protect and preserve nature. This collaborative effort managed to strengthen the bonds among participating teams through a meaningful group activity. As iGEM uniCRETE, we chose to plant an olive tree given our signature involvement with olive production during the making of DeltaSense.

  

• iGEM ABOA’s World Environment Day Collaboration Teams from all over the world made a small video, each one talking about protecting a different part of nature. Our team decided to collaborate by using the phrase : “Let’s save our flora!”, once again paying tribute to the olive tree.
  

  
  
  

  Participating in these diverse and inspiring collaborations was indeed a privilege. It allowed us to create closer connections with fellow iGEM teams while also providing a platform to share our love for science. These experiences enhanced the power of unity and collaboration in advancing our shared pursuits in the realm of synthetic biology and beyond.

SynBioCrete Chronicles blog: Nurturing Synthetic Biology Education

One integral component of our social media and communication endeavors has been the establishment of "SynBioCrete Chronicles," our dedicated online blog. Within this platform, we delve deeper into the realm of synthetic biology and its related fields, uploading self-created articles with a strong educational focus. Simultaneously, our blog served as a platform to explain our project, "DeltaSense" to a broader audience. Additionally, we explored a diverse array of subjects including ethical considerations in bioscience research, a topic we certainly consider worthy of further discussion. SynBioCrete Chronicles played a vital role in informing individuals who may not have previously explored the wonders of science, helping them become more knowledgeable while also entertaining them. In a nutshell, our blog embodies our mission to make synthetic biology education inclusive and inspire our society to explore the boundless possibilities of this field.

Exploring Bioethics: A Multidisciplinary Open Discussion on Environmental Responsibility and Human Prosperity

Exchange of ideas has always been a powerful tool for us through all stages of making DeltaSense, from initial brainstorming to the everlasting process of questioning our methods and results to achieve the maximum impact of our project. In this spirit, we designed and conducted an open discussion through a zoom panel, where invited experts and students of various fields had the chance to share their views on the matter of environment exploitation and human prosperity.

Specifically, we facilitated a conversation involving Dr Moschou, a distinguished professor of molecular plant biology at the University of Crete, Mr. Polychronides, a political scientist and rhetoric coach, Mr Kalafatas, a Chemistry student and Mr. Tsarikoglou, a Chemical Engineering student. Before the event, we assigned the participants bibliography correlated to bioethical matters such as the Tragedy of Commons and Environmental Kuznet’s Curve and incentivized our esteemed speakers to present case studies where human intervention had an impact on the environment. During the event, we were mesmerized by the common lines drawn between ecology, politics, economics and manufacture through strong yet polite argumentation. We introduced, to the energetic audience, terms like rewilding and restoring versus the traditional concept of sustaining environmental resources and discussed how attribution of charges to companies violating environmental protection legislation is a top priority. Broadly, the role of education in forming environmental consciousness was highlighted by the participants and inspired us to take action as iGEM UniCRETE and train young people to be more sensitive as far as bioethical matters are concerned.

Sprouting Ethics: Unveiling Bioethics through “The Farmer of the Green Olive Grove” Book Presentation and Engaging Activities with Young Minds

It is no secret that teaching ethics at a young age lays the groundwork for responsible decision-making and empathetic awareness. Bioethics, a branch of ethics focused on life sciences and healthcare, can also be introduced gradually so that early exposure to bioethical principles fosters a sense of responsibility towards the natural world. Given our purpose to give young learners a solid foundation in environmental responsibility, we visited a primary school in Heraklion,Crete, and presented a book we crafted to break down the complexities of pesticide use, titled : “The Farmer of the Green Olive Grove: Adventures in Organic Farming and Biotechnology”. Through this simple story, young readers could discover farmer Giannis’ commitment to organic farming and the use of synthetic biology and DeltaSense to protect his olive grove from pesticides. Tailoring this important concept to a child’s level of understanding meant the inclusion of vivid illustrations and simple dialogues within the demonstrated manual, as well as engaging storytelling.

During this session, we shared stories brought to life by our compass book and prompted conversations about pesticide use, its effects, and the ethical questions it raises. We then engaged in interactive activities such as roleplaying to trigger the children’s creativity and critical thinking into coming up with alternative endings to the story we presented. Despite the Greek school system's restrictions on access to such topics, we were pleasantly surprised by the young students' productive curiosity and insightful questions. Overall, our visit successfully introduced a novel perspective on bioethics and environmental consciousness among young but surprisingly bright minds. Upon our departure, we donated sufficient copies of our book to the school’s library for our young friends to borrow.

Distribution of Questionnaires in Collaboration with the Cretan Association of Organic Farmers

Our team wished to communicate the purpose of our mission with people who would be directly affected by our project and raise their awareness. From the beginning of our project, we had in mind that farmers had a distinguished position as people we wanted to contact, inform and get feedback from. Aiming to make conclusions about the lack of knowledge of the differences between regular and organic cultivation and the use of pesticides, we came in contact with the Cretan Association of Organic Farmers. The association expressed their interest, thus, we provided them with questionnaires to fill out and to hand out to their customers and we concluded the following results.

I believe that the increased price of organic products is a deterrent factor for their purchase.

Are you informed about the danger of the excessive use of pesticides?

Do you think that the overuse of pesticides harms the overall health?

Active Participation in European Researchers' Night at the Foundation for Research and Technology Hellas

The "Researchers' Night" has evolved into a prominent pan-European celebration of science and technology, initially introduced by the European Commission about 15 years ago. The Foundation for Research and Technology-Hellas (FORTH) has been a trailblazer in Greece and Europe, securing EU funding to organize Researchers' Night events since 2005. Its main objective is to address the growing need for academic and research institutions and teams to engage in large-scale outreach activities and connect with local communities. Over 370 European cities, including Heraklion, Crete, participate, showcasing the work of researchers and fostering public familiarity with the world of research. Our team could not be absent from such an event aimed at reducing the gap between the ordinary (scientific and not) audience with the biosensing system DeltaSense. The participation of our team with its poster stand and bench was of great importance in explaining to people of all generations our vision for the environmental future of Crete. In order to achieve this, there was a series of three activities aimed at visitors. The initial phase involved a presentation by our team, providing insights into the competition, our project, and the overall concept. During this segment, the audience gained an understanding of the issue we are addressing: excessive pesticide use, specifically targeting deltamethrin. Through significant statistics, we elucidated the toxicity of deltamethrin, discussing its applications, concentrations, and potential penetration into aquifers. Concurrently, we introduced a schematic representation of our biosensor system. The second task entailed a 3D reconstruction of the biosensor materials, including DNAs, enzymes, and magnetic beads, using small colored sticks and modeling clay. This interactive session allowed both children and adults to understand the binding between the aptamer and deltamethrin, comprehend the sequential hybridizations, observe the role of magnetic beads, and understand the final visualization system involving lipase. Spectators could follow the process from one step to another by connecting the three-dimensional sticks. Simultaneously, to explain the action of lipase and suppress the visual signal given by the biosensor, we demonstrated tubes with colored water indicating high concentration of deltamethrin and others containing pure colorless water signifying deltamethrin’s absence.This visually stimulating activity allowed us to present how our aptamer biosensor works to common people. Although the system we present can be applied in many cases with an appropriate change of aptamers and the corresponding change of sample, the detection of deltamethrin in water is aimed at our vigilance for environmental purposes and our commitment to environmental legislation. That is why at the end of our presentation we asked people we interacted with to write or draw a thought about the project and a habit they would like to adopt from now on to protect the environment. By gathering a large number of such thoughts we finally created a coliseum, the so-called ‘’Wall of Promises’’.

Public Engagement with Farmers and Customers of our Local Farmer Market

When we processed the results from the survey we conducted in collaboration with the Cretan Association of Organic Farmers, we decided to take matters into our own hands. We visited the farmer market of Kamminia region in Heraklion Crete to talk about the problem of pesticides’ overuse and our mission to abridge it. We discussed with several farmers and the majority of them shared their concern about the difficulties and insecurities of their occupation due to the lack of governmental financial support. They complained about the absence of a prevention and treatment governmental plan and emphasized its importance as it can save their production and subsequently their earnings in case of a parasite outbreak. When we presented them DeltaSense to them they expressed their enthusiasm and positiveness in buying it if it would reach the market’s selves. Organic farmers were particularly keen on our project as it would help them maintain the strict pesticide concentration limits mandated by the European Union for organic certification. At the same time, traditional farmers recognized the importance of adhering to these limits to ensure the safety of their produce. The overwhelming support from the farmers at the market left us feeling motivated and optimistic about the potential success of our project in the agricultural community. Finally, we asked farmers and customers the following question in order to extract data regarding the acceptance of DeltaSense from the public.

If you had a tool that could detect pesticides in agricultural products and you could find out that an organic product has close to zero concentration of pesticides and a regular one of the same kind has pesticide concentration close to the permitted limit, which one would you buy?

Implementation

Introduction

Equipped with a wealth of knowledge and diverse perspectives for potential expansions and enhancements of our project link gia integrated, our team collectively made the decision to thoroughly delve into all the suggestions we gathered from our discussions with academics and individuals, employed in the agricultural and business sectors. Our aim is to transform DeltaSense from a theoretical concept into a tangible and innovative detection method, ensuring its practical existence and effectiveness. Ιn order to achieve this purpose we decided to frame the implementation of the project around four main axes

1. Which samples can we use the system for, and how can we obtain them? What other adaptations can be done?
2. How could the biosensor be even more accessible? What could be the anticipated cost?
3. Who are the possible end-users?

1. Which samples can we use the system for, and how can we obtain them?

In the initial phase, our emphasis on the first axis was on exploring proposals that had been previously voiced and thoroughly deliberated by experts in the field. To establish a connection between theoretical concepts and practical applications, and to align the straightforward implementation of DeltaSense with the transformation of detection practices in the field, the team conducted an extensive literature search. Subsequently, we pinpointed protocols for processing samples originating from various potential sources of deltamethrin. Our endeavor involved modifying the samples to a finalized state that could be effectively detected for pesticide residues using our biosensor.

1. Determination of deltamethrin in water samples

A volume of 100 mL runoff water is collected from different agriculture fields where deltamethrin has been sprayed. The samples are filtered and 1.0 mL of 5.0% EDTA is added to each of them to remove various metal ions. Each sample is extracted with chloroform (2x 100 mL). The extracts were combined and washed with 20 mL of 0.1 M K2CO3 solution to break any emulsions. The chloroform extracts were dried over Na2SO4 in a filter funnel and the filtrate is collected in a 250 mL calibrated flask. The filter funnel is washed with 20 mL of chloroform until the volume of the filtrate is made up in the mark. known aliquots of chloroform extracts were taken under reduced pressure. The residue is dissolved in 10 mL methanol.

2. Determination of Deltamethrin in soil sample

Several studies have shown that the residues of pesticides in the soil is a key factor for food contamination and biological chains. As Mr. Fafoutakis (multichrom.lab) claimed, it is important to check whether there are residues of deltamethrin in the soil and to see if the quantity exceeds the permissible limits. Dispersive liquid–liquid microextraction procedure is applied so as to extract deltamethrin from the samples and be further tracked.
More specifically, the procedure shall include:
In a 2.5 g well-powdered soil sample, deltamethrin (if existing) is extracted with 25 mL (55mL) methanol, then filtered using Whitman filter paper and made up to 25mL with the same solvent.
Even though deltamethrin is frequently employed in olive groves to manage downy mildew, resulting in environmental degradation and disruption of the ecological balance, its application is not confined solely to that context, as Prof. Vontas underlined. Pyrethroid pesticides, as they are also widely used on crops like cotton, fruits, rice, and wheat, appear to be a significant source of sediment toxicity in urban and agriculturally dominated streams also there. In the case of detecting deltamethrin in these samples, beyond the environmental dimension of the issue, a serious problem of food safety and public health arises.That is why, these crops can also be collected as a sample for analysis.
More specifically, the procedure shall include:

3. Determination of deltamethrin in vegetables and fruits

25 g of vegetable or fruit samples are collected from agricultural fields, where deltamethrin has been sprayed as an insecticide. The samples are macerated with 2× 20 mL EtOH-d.H2O (1 : 1), filtered through a thin cotton cloth and the filtrate is centrifuged. The filtrate is quantitatively transferred into a 25 mL volumetric flask and made up to the mark with 50% ethanol. Aliquots of supernatant are taken in a 25 mL graduated cylinder and then analyzed. Filtrate from foliages are passed through a silica gel column filled with 5.0 mg silica gel, which is found to be sufficient for removal of chlorophyll and other interfering materials present in the extracted sample. The column is washed with 10 mL of 50% ethanol, washings are collected in a 25 mL volumetric flask and further analyzed.

4. Determination of deltamethrin in grain samples (rice and wheat)

Different samples of grains (rice and wheat) (25 g) are collected from the fields where deltamethrin has been sprayed. The samples are weighed, macerated and blended in a mixer. Then 1.0 mL of 5.0% EDTA is added to the blended sample and is extracted using 25 mL chloroform. The chloroform solution was then decanted into a 250 mL calibrated flask through Whatman No. 1 filter paper. Blending and decanting is repeated 2×10 mL chloroform. The extracts are combined and diluted to the mark. The chloroform extract is evaporated off, under reduced pressure. The residue is dissolved in 10 mL methanol.
In the cultivation of olive groves, the application of insecticides and herbicides is vital for crop protection and enhanced production, as we have already mentioned in the Project Description. The trend of surpassing established limits concerning especially the presence of deltamethrin residues in olives and resulting olive oil poses potential risks to human health and it is associated with adverse effects, including acute toxicity and long-term health risks. Given that olive oil extraction involves mild fermentations, any lipophilic pesticide residues from the groves can contribute to chemical contamination in the final product. With an average production ratio of 4 kg of olives yielding 1 kilogram of olive oil, there is an urgent need for efficient controls to ensure that residual levels in the oil adhere to permissible Maximum Residue Limits (MRLs). Our biosensor can be expanded to assess oil samples, incorporating an initial liquid-liquid extraction process to extract a portion of the pesticide for thorough analysis.

More specifically, the procedure shall include:



C. Determination of deltamethrin in vegetables and fruits

25 g of vegetable or fruit samples are collected from agricultural fields, where deltamethrin has been sprayed as an insecticide. The samples are macerated with 2× 20 mL EtOH-d.H2O (1 : 1), filtered through a thin cotton cloth and the filtrate is centrifuged. The filtrate is quantitatively transferred into a 25 mL volumetric flask and made up to the mark with 50% ethanol. Aliquots of supernatant are taken in a 25 mL graduated cylinder and then analyzed. Filtrate from foliages are passed through a silica gel column filled with 5.0 mg silica gel, which is found to be sufficient for removal of chlorophyll and other interfering materials present in the extracted sample. The column is washed with 10 mL of 50% ethanol, washings are collected in a 25 mL volumetric flask and further analyzed.

D. Determination of deltamethrin in grain samples (rice and wheat)

Different samples of grains (rice and wheat) (25 g) are collected from the fields where deltamethrin has been sprayed. The samples are weighed, macerated and blended in a mixer. Then 1.0 mL of 5.0% EDTA is added to the blended sample and is extracted using 25 mL chloroform. The chloroform solution was then decanted into a 250 mL calibrated flask through Whatman No. 1 filter paper. Blending and decanting is repeated 2×10 mL chloroform. The extracts are combined and diluted to the mark. The chloroform extract is evaporated off, under reduced pressure. The residue is dissolved in 10 mL methanol.

In the cultivation of olive groves, the application of insecticides and herbicides is vital for crop protection and enhanced production, as we have already mentioned in the Project Description. The trend of surpassing established limits concerning especially the presence of deltamethrin residues in olives and resulting olive oil poses potential risks to human health and it is associated with adverse effects, including acute toxicity and long-term health risks. Given that olive oil extraction involves mild fermentations, any lipophilic pesticide residues from the groves can contribute to chemical contamination in the final product. With an average production ratio of 4 kg of olives yielding 1 kilogram of olive oil, there is an urgent need for efficient controls to ensure that residual levels in the oil adhere to permissible Maximum Residue Limits (MRLs). Our biosensor can be expanded to assess oil samples, incorporating an initial liquid-liquid extraction process to extract a portion of the pesticide for thorough analysis.

More specifically, the procedure shall include:

E. Determination of Deltamethrin in Olive Oil Samples

• Liquid-Liquid extraction 1:

An aliquot of 0.001 g of olive oil is weighted in a 40 mL screw-capped glass tube and dissolved in 5 mL of n-hexane. The solution is extracted twice with 10 mL of ACN.

• Liquid-Liquid extraction 2:

An aliquot of 0.001 g of olive oil is weighted in a 40 mL screw-capped glass tube and dissolved in 5 mL of saturated n-hexane in ACN. The solution is extracted twice with 10 mL of ACN saturated in n-hexane.

5. Liquid-Liquid extraction 3:

An aliquot of 0.001 g of olive oil was extracted twice with 10 mL of ACN in a 40 mL screw-capped glass tube. Each extraction test was performed by agitation in a rotary shaker for 5 min. The combined extracts from each procedure were brought to dryness under reduced pressure.

In all the above processes, the extracts from the whole extraction procedure are concentrated under reduced pressure and then the residue is redissolved in methanol or in acetonitrile. Both solvents are polar organic solvents that due to their increased polarity, are miscible with water. Therefore, by dissolving the residue in an initial amount of polar organic solvent, we can dilute it further in water, thus making an aqueous solution in which our biosensor can function exactly as in other aqueous solutions of deltamethrin, as the artificial solutions mentioned. In conclusion, we find that regardless of the original sample, such as oil, soil and fruit, a process of recovery of deltamethrin can be implemented to extract deltamethrin and prepare aqueous solutions. Although aqueous, it is required to contain some polar organic solvent to make deltamethrin's dissolution possible. The subsequent transformation of the sample into its liquid form, facilitating the application of the biosensor, confirms its versatility and adaptability as a deltamethrin detection method. Essentially, the innovation of our team in the contemporary market of detection methods focuses on the broader dissemination of knowledge and technology related to the cell-free aptamer biosensor system. This model has the potential for various applications due to its exceptional selectivity. Additionally, this innovative aptamer-based biosensor system can be widely used by adapting only the aptamer piece to the new compound we want to detect, whose determination is automatically possible. During the discussions with experts in the field of agricultural science and analytical science, they emphasized the importance of developing such a tool that, with only a single step, can be adapted to a new need that arises at an environmental level. As it is direct and sensitive, with further improvements, it can be implemented and flexible to meet emerging needs for the detection of the desired substances.

2. How could the biosensor be even more accessible? What could be the anticipated cost?

The accessibility of our biosensor is linked to both the concepts of portability and affordability. Regarding the former, as emphasized by Mr. Garinis and Mrs. Gkizeli, the project can take on a much more business-oriented and marketable aspect if presented to the consumer audience as an easy-to-use, portable kit. This automatically transforms it into a much more accessible solution. A portable biosensor offers multiple advantages compared to a non-portable one. For instance, it can be stored and used whenever the user wants to. Additionally, it allows the on-site analysis of the sample which is quite useful especially when the transportation of the sample to the laboratory may affect the sample’s condition and subsequently, the results of the analysis. In a reasonable timeframe and with the help of a few tweaks and reactions, without the intervention of high-tech expertise, it can provide the desired result for the presence or absence of deltamethrin. That way of thinking led our Wet Lab to search about the way of creating a portable, easy to use, biosensing kit for deltamethrin detection in water samples. As we learnt, a commonly applied tactic to produce such portable biosensing kits is the lyophilization of all the required reagents. Lyophilization, also known as Freeze-drying, is a process in which water in the form of ice under low pressure is removed from the reagents by sublimation. There are 3 remarkable ways in which lyophilization could allow the production of a portable biosensing kit. Firstly, by the removal of water the half-life of all biomolecules, such as the DNA molecules and proteins used in our system, will be significantly extended as many reactions that could potentially cause their degradation need water solution to take place. Secondly, molecules in a solid dried state are way more resistant to thermal denaturation and therefore can preserve their biological properties and function more easily. Finally, a dry environment inhibits the growth of many microorganisms, such as bacteria and fungi, that could potentially damage biomolecules’ integrity. It can now be easily understood that lyophilized molecules are way more damage resistant during handling, transportation, and storage and they are significantly useful during the making of a portable biosensing kit.

There are several lyophilization protocols specific for the freeze-drying of the desired biomolecule. In the case of proteins, two of the most common are the “Lab-Scale Lyophilization on Reverse-Phase Fractions From High-Performance Liquid Chromatography Run” and “Pilot Scale Freeze-Drying”. Despite their differences, all the protocols of protein lyophilization share some very important reagents and steps. First of all, it is absolutely necessary to use a buffer solution to maintain the pH at the desired levels for the preservation of protein stability and structure. The most preferable ones are histidine, citrate and Tris buffer solutions that prevent the alteration of pH during the Freezing. Additionally, all of the aforementioned protocols use some kind of stabilizer molecules, such as trehalose, sucrose, mannitol and glycine which are hydrogen bond-forming organic molecules that play a crucial role in the preservation of the protein’s structure during the freezing. Continuing, all of the lyophilization experiments are conducted in the same general steps. First of all, there is a freezing step during which the protein solution is freezed inside a general laboratory glassware item such as glass flask, glass vials and other. Lastly, there is the drying process in which the freezed solution is placed inside a temperature controlled condenser at -60oC and 10-100μbar.

The freeze-drying of DNA molecules also follows the same roles. The presence of a buffer solution and stabilizing molecules such as sucrose and D-mannitol is necessary. As described by K. Jung et al., 2020 a freezing step at -80oC and an overnight drying step at -85oC and 0.04mbar pressure are sufficient for lyophilizating a DNA-based biosensor such as ROSALIND. In regards to the portable kit, our biosensing system would consist of 3 separate eppendorf tubes which contain the lyophilized dAPT, L1 and L2 molecules (tube 1), Cas12a with its crRNA (tube 2) and Lipase substrate p-nitrophenyl ester (tube 3), respectively. It is noteworthy that a Cas12a/crRNA complex can in fact be lyophilized as the work of Grant A. Rybnicky et al., 2022 indicates. Additionally, it would have another eppendorf tube (tube 4) that contains in a water solution the SA-MBs-LINKER-Lipase complex. Last but not least, it would have a magnet which is a necessary component for the correct conduction of the biosensing system.

During the testing, the user would add the examination sample inside tube 1. This would firstly, rehydrate the DNA molecules but also would allow the binding of deltamethrin to dAPT and the subsequent L1-L2 duplex creation, as described in the Engineering Success Section. Afterwards, the user will add to the sample, the Cas12a-crRNA complex which is in powder in tube 2. The mixture will then be transferred inside tube 4 containing the SA-MBs-LINKER-Lipase complex thus allowing the interaction between Cas12a and LINKER, and in the case of Deltamethrin’s presence, the subsequent hydrolysis of LINKER. The tube 4 will be placed on top of a magnet causing the localization of the magnetic beads at the bottom of the tube. As described previously, if the water sample contains deltamethrin, Lipase will be left in the supernatant, otherwise it will be localized at the bottom of the tube. In the final step, the user will pour the solution contained in tube 4, while keeping its bottom in contact with the magnet, into tube 3 which contains Lipase’s substrate. In this way, only the supernatant of the mixture will be transferred to the new tube but the magnetic beads and possibly Lipase will remain at the bottom of tube 3. Lastly, the user would finally be able to see whether deltamethrin was present in the primary sample collected from the field by observing the color change (or not) of the last solution.

For the estimation of costs of the detection system we developed, we consulted Mrs. Gizeli as she is actively involved in the business part, especially in the field of biosensors. In this discussion she explained to us an initial approach to the costing of a system. In particular, she said that at this early stage of the development of the biosensor the costing is based exclusively on the materials required for its operation. It does not include the cost of personnel as this is a subsequent stage of the approach. That is, the initial stage is the cost of the reagents. Afterwards, when the sensor is fully developed and in the launch stage, the way of approaching the price changes as in mass production the cost is further reduced due to the reduced price of reagents. More specifically, when companies are asked to bid for reagents, large quantities have a lower cost per mg of reagent compared to smaller quantity packages. So, after discussing the way in which an initial approximate price is arrived at, we then estimated the price for a biosensor.

The procedure we followed was to look at the prices of all the orders we made, and reduce the quantity we bought to the quantity required for a single biosensor. This way, we can find the price of each reagent for the quantity required for a single biosensor. This procedure was carried out for all the reagents used and the cost came out to about 10 euros, i.e. for an initial stage quite low. This price makes our biosensor accessible not only to national organizations carrying out controls and analyses on samples, which can dispose of a large sum of money, but also to smaller laboratories and even to the farmer who wants to make sure that the spraying has not exceeded the legal limits.

Certainly, as we have already mentioned, there are different analytical methods for determining the concentration of different substances in samples and indeed they are very reliable methods, such as liquid chromatography, gas chromatography, mass spectrometry. However, the above methods are very expensive, not only in terms of consumables required for the analyses but also in terms of equipment, the price of which is quite high and requires frequent repairs and maintenance. Furthermore, these analytical methods are time consuming and technical expertise is necessary since the complexity of the method. Instead, our novel biosensor requires no expertise. So, the possible initial price of our biosensor is affordable for everyone to use.

3. Who are the possible end-users?

One aptamer-based biosensor for detecting deltamethrin in water samples aims at preserving ecological balance and conserving Cretan heritage through a broader adherence to the regulations governing the use of this particular pyrethroid. The end-users of the biosensor can be diagnostic laboratories and institutes that routinely analyze dozens of samples daily. As mentioned by Mr. Tsigos and Prof. Spilianakis, existing detection methods require expensive equipment and training. Additionally, according to Mr. Manikis, the biosensor can gain commercial significance at the level it targets, addressing the producer who would prefer an easy, quick, and straightforward testing of their samples. The Delta Sense comes as a solution to this desire.

References:

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