Police and Criminology

Finnish forensic chemists

After deciding on the topic for this year’s project, we reached out to Finnish forensic chemists to get the first reviews and comments on our idea. At their request, we will refer to them simply as forensic chemists, without naming their organization or individual experts we have talked with.

During the meeting on March 26, we presented the current progress of our research, outlined the project idea, and discussed its potential benefits for crime scene investigations. We learned that the most accurate and widely used analysis method in Finnish crime investigations is currently DNA profiling. However, collecting and analyzing both relevant and potentially irrelevant samples for DNA testing takes time and resources, which may delay justice. This remains one of the challenges in pretrial investigations that we aim to address.

Experts’ feedback guided us in choosing between the two directions we had in mind for our project. We had considered either trying to determine the number of days since the blood was bled, or identifying the time of day based on the circadian rhythm by measuring melatonin and cortisol levels in the blood. Experts commented: “If you need to prioritize, we would encourage you to work on determining the age of the bloodstains in days/weeks first.” (More details on how we selected our topic can be found in the project description part of the wiki.)

We discovered that there is currently no established or reliable method in use for determining the age of bloodstains even though there has been efforts to make such a test. Instead, investigators often have to rely on visible color changes of the bloodstains and witness statements to estimate when the crime occurred. This approach is subjective and can lead to uncertainty about crucial evidence.

This discussion gave us confidence that developing a test for estimating bloodstain age would fill an important gap in forensic investigation techniques. Initially, we were concerned about the accuracy of our proposed method, since we found a study [1] where it was shown that after 2 weeks no Human Serum Albumin was found in bloodstains. Using that study we made a hypothesis that our test can generally indicate whether a bloodstain is older or younger than about two weeks. Ideally, we would want to create a test that can estimate the exact number of days since the blood was bled. However, the experts assured us that even an indicative test would be very useful. During the early stages of an investigation it is especially critical to build a broader picture of how the crime occurred. Based on this feedback, we decided to prioritize developing a test that could differentiate bloodstain age on a days-to-weeks scale, with the goal of later improving it to estimate more precise timing.

At the end of our meeting, the experts also emphasized some important practical requirements. The test should be simple enough for use directly at the crime scene by investigators or police officers. It must be cost-effective, as police departments have limited budgets and equipment. Margins of error and other limitations to the test should be considered in-depth.

Taking all this into account, we adapted our project design to develop a portable, on-site test that uses only a small sample amount, gives an easily analyzable bioluminescent signal, and remains affordable. We also ensured that our planned sample handling process uses DNA-free instruments and our test is cell free to avoid contaminating the crime scene.

Senior Constable Daniel Kalejaiye

On June 13th, we met with Senior Constable Daniel Kalejaiye, also known as Konstaapeli Daniel. He is a senior constable and an internet police officer known for his social media content aimed at children and youth. His social media work focuses on making police-related topics understandable and accessible to younger audiences.

We began by discussing how to best communicate our project to the public. Given the serious nature of our topic, we were concerned about unintentionally upsetting people or miscommunicating key points. We were especially worried about how we should approach communicating about blood as it can be a sensitive topic and is censored in some countries. Kalejaiye reassured us that as long as blood is presented as a neutral part of our project, it should not raise concerns. He suggested using illustrative ways, such as ketchup or a red marker, to demonstrate blood without risks for discomfort.

He also emphasized the importance of transparency to reduce public suspicion and ensure clear communication. For example, he encouraged us to openly state that our project is still in the research phase and that we will not have a finalized product by the end of this year’s iGEM. This has been a common misconception among stakeholders and one that can be proactively addressed.

To ensure clarity in public engagement, Kalejaiye advised against including too much information in a single social media post. Instead, he recommended tailoring communication to the youngest segment of our target audience, stating: “If they can understand the concept, older audiences likely will too.” This strategy helps ensure our messaging is both accessible and easily digestible. He also encouraged us to be creative, enthusiastic, and to use familiar analogies to make our project more relatable to the public.

Inspired by his advice, we created a cartoon style illustration of the molecular mechanism of our project. Our goal was to simplify scientific concepts behind our test in hopes of demystifying our project as a whole and prevent potential misunderstandings.

We also explored how police might use a tool like ours and what requirements they might have for it. Kalejaiye explained that police training prioritizes the practical use of tools rather than the theory behind them. Police officers are generally not familiar with the operating principles behind their equipment, but they must understand how to use it reliably and recognize its limitations. Kalejaiye emphasized that our test should be simple and intuitive to use, which aligned with what we had already considered during the design phase.

We learned that police officers are trained to use the most common tools during their basic education, and that there are additional courses available for newer or less common tools such as ours. However, these training sessions are often based on broader themes rather than specific equipment. Kalejaiye suggested that a simple bullet point structured training packet would likely be sufficient for introducing our test in a training context. A more detailed explanation should be saved for the National Bureau of Investigation assessing our test.

Overall, our meeting gave us valuable insights into both police work and effective communication. Kalejaiye’s enthusiasm for our project was highly encouraging and inspired us to approach our outreach efforts with the same level of passion and clarity.

Senior Detective Constable Samuli Lehikoinen

On June 17th, we had the chance to meet with Senior Detective Constable Samuli Lehikoinen, an experienced crime scene investigator from the Southwest Finland Police Department. Lehikoinen, with over 24 years in the police force and 12 years specializing in crime scene investigations, shared valuable insights into forensic practice, especially relating to bloodstain pattern analysis. He’s been trained internationally, most notably in the Netherlands, which is a renowned forensic hub in Europe, and he regularly participates in investigations focusing on bloodstain evidence.

The aim of our meeting was to understand how our bloodstain age determination test could be integrated into actual crime scene investigations, including its practical applications, potential obstacles, and the real-world conditions it would face. Furthermore, we were also curious about what other methods are performed on bloodstains at the crime scene.

Lehikoinen immediately highlighted the practical challenges our project faces: blood drying and aging processes are influenced by numerous variables, such as temperature and humidity. Nevertheless, he saw real potential in our test, especially if we can achieve accuracy within a one-day time frame. That level of precision would already make a significant advancement in the field of crime scene investigation as it would provide a method for determining the age of a bloodstain, which is currently impossible. Moreover, we were informed that maintaining confidentiality is not typically difficult, as most crime scenes are located in private residences.

Currently, crime scene investigators are able to determine the area of origin from the spatter stains. The technology has the capacity to reduce the origin area to the size of a football as well as determine the victim’s position at the time of attack and it could give a preliminary indication of what type of weapon was used. Furthermore, it came to our attention that there are chemicals capable of restoring bloodstains, if they have been washed away from the crime scene.

Lehikoinen also clarified the usual course of action during an investigation. First, the first patrol at the scene assesses the situation and consults with crime scene investigators by phone. The decision is made by the latter as to whether it needs to be investigated by crime scene investigators. In cases of suicide by shooting, some samples are recorded by the first patrol at the scene. In contrast to other countries, crime scene investigators are able to conduct their investigations independently, without the need for a medical examiner. Typically, two crime scene investigators are responsible for the scene, including conducting investigations, documenting samples and cordoning off the area. However, it should be noted that each case is unique and the procedure may vary.

Currently, crime scene investigators rely heavily on indirect clues to establish timelines, such as a victim’s body temperature, decomposition state, or even mundane details like incoming mail and phone activity. Bloodstains alone are currently considered unreliable for precise time determination due to environmental variability. However, our test could offer clear added value by objectively distinguishing recent bloodstains from older ones, allowing investigators to quickly focus on relevant evidence.

Lehikoinen emphasised that crime scenes, particularly indoors, often present fairly stable conditions, usually around 20 °C. Thus, we assume that our test would be effective for typical Finnish violent crimes, which often involve alcohol, jealousy or sudden aggression at private residences. We were told that it would be particularly useful in cases of unclear death, in homicides and in other violent crime cases. We are verifying the oxidation of albumin in a laboratory setting by aging bloodstains. However, in outdoor settings, the reliability of our test would be compromised if we would be unable to identify clear margins of error. Unfortunately, we don’t have the resources to conduct any tests outside the laboratory, but we would definitely do so if it were possible.

We also learned about practical aspects of field investigations, such as the importance of simplicity of forensic tools. Investigators deal with a wide range of devices and methodologies, and introducing complicated, rarely used tests can be counterproductive. A straightforward design, that has few components and does not require special appliances, would maximize the usability of our test.

Another important insight involved the validation and implementation processes of new forensic tools. Despite minimal legislative barriers, extensive validation and local verification are essential before new methods are widely adopted. Lehikoinen made it clear that funding and bureaucratic implementation processes are common bottlenecks, and thus cost-effectiveness and clear added value are critical for any new technology.

Lehikoinen also emphasised the importance of simplicity, practicality and presentation of our test, given the limited time available to crime scene investigators to attend training courses. In addition, we learned that most specialized courses are held abroad, and that crime scene investigators apply to these courses themselves. In conclusion, this conversation had a significant impact on our understanding of real-world application of our test.

Doctoral Researcher Mari Humalajoki

June 2025
On June 23rd, we met Mari Humalajoki, who holds a double master’s degree in forensic science from Uppsala University and in molecular genetics from the University of Helsinki, and is now pursuing her PhD at the University of Helsinki. With an extensive background in genetics and forensic science, including hands-on research experience at Finland’s National Bureau of Investigation, we were offered insights into forensic methodology and realities of implementing new forensic tests. We got in contact with Humalajoki thanks to a member of the Aalto-Helsinki iGEM team, with whom we collaborate. This person introduced us and our project to Humalajoki.

Forensic research in Finland is typically restricted to the labs of the National Bureau of Investigation, meaning external methods are rarely adopted without thorough internal validation. Despite standardized forensic protocols, research findings are often not shared outside of official channels, limiting public awareness and external scientific contribution. We received positive feedback regarding our university based forensic research and were encouraged to clearly position our test as complementary, rather than competitive, to increase the likelihood of its acceptance into established workflows.

One critical takeaway was the practicality and significance of nondestructive methods. We had initially formed the impression that at crime scenes there is always plenty of blood based on our previous meetings with experts. However, we were reminded that people might try to clean the crime scene after the crime. We were also told that destructive testing, such as DNA extraction from small or limited samples, is carried out cautiously and sparingly.

A method like ours, which preserves evidence integrity, would be extremely valuable, especially if it does not interfere with standard forensic chemicals. For instance, luminol, widely used to detect bloodstains, is considered harmless for downstream DNA analysis but could interfere with some analytical tests due to its fluorescence. Humalajoki stressed that if our product can reliably function after luminol use, it would significantly enhance its appeal and usability. After meeting with Linda Wager and Senior Constable Anu Vihavainen we received the confirmation that luminol would be used after our test.

We were also advised to pursue practical experimental setups to demonstrate proof of concept clearly and robustly. Tests, such as exposing blood samples to UV radiation, humidity or sunlight for varying periods, would help illustrate the reliability and limitations of our approach. However, we lack the resources, such as appropriate spaces, to conduct these tests in the laboratory, so instead we looked more closely into what advice we could receive from other experts. Interestingly, even up to a margin of error of 30% in measurements can be acceptable in forensic contexts if adequately documented and understood. Additionally, promising areas within forensic genetics, such as epigenetic markers and RNA profiling, were introduced to us. These indicate future possibilities for our research beyond the current focus on albumin oxidation.

During our discussion, we also learned that popular forensic media is generally accurate and suitable for educational purposes, however dramatized content such as CSI often lacks real life counterparts. For example, we were recommended videos from WIRED’s forensic investigator, Matthew Steiner. With this knowledge, we now have the opportunity to learn more about forensic procedures ourselves.

Overall, our conversation with Mari Humalajoki provided us with valuable guidance toward a carefully validated, nondestructive, workflow compatible forensic tool that would hold substantial value for crime scene investigators, not only in Finland but internationally. We kept in touch with Humalajoki after the meeting, updating her on our project.



September 2025
On September 10th, we met again with Mari Humalajoki to discuss our latest results and explore potential pathways for expanding our work to contribute meaningfully to future projects in forensic science. The discussion remained firmly grounded in practical considerations and empirical data. She noted that our Nb77 + ALB8 readouts were great and show the expected decreasing trend in relation to bloodstain age (link to results) and a pronounced drop around days 15-20 that tracks published findings by K. Rajamannar [1].

We also discussed the criteria for the adoption of a new forensic science method into the Finnish criminal investigation system. There are no dedicated institutes for forensic science technology in Finland, which might be the reason why there is very little research on forensic science related topics in Finland. We were told that, a university group like ours can add value by conducting preliminary research and communicating its findings in a clear and concise manner. In that regard, she commended the clarity and goal-oriented tone for our wiki page and encouraged us to continue highlighting our test’s strengths and limitations in precise and accessible language.

We were proposed a straightforward approach to categorizing our stakeholders: attorneys, who depend on precedents; investigators, who face strict time constraints and scientists, who are responsible for translating evidence into a format that courts can accept. Across these groups, the same principle applies. Forensic tools are more effective when they are quantitative, minimize interpretation, and are easy and inexpensive to use.

Linda Wager and Senior Constable Anu Vihavainen

During our discussions on June 17th with police officer Samuli Lehikoinen, he recommended us to also consult with the Finnish police quality manager. Following his advice, we reached out to Linda Wager, who serves as the quality manager for the Eastern Uusimaa Police Department and has previously worked as a crime chemist at the Finnish National Bureau of Investigation. To further enrich the discussion, Wager invited Senior Constable Anu Vihavainen, a police officer and specialist in blood trail investigations, to join our meeting.

On July 9th we met with the experts and were pleased to receive encouraging feedback on our project idea, along with confirmation that such a tool is indeed needed in forensic work. Vihavainen, who has over 12 years of experience in crime investigations, emphasized that our proposed test could be essential for identifying which bloodstains are truly relevant for more detailed analyses. She explained that this would be particularly valuable in cases of the complex crime scenes, like the living spaces where multiple bloodstains of different ages might be present. For instance, she described situations where it is impossible to rule out, with objective evidence, if suspects' blood found on a crime scene is from that case or, for example, from a nosebleed a year ago. In such cases, there is currently no reliable method to determine which bloodstains are relevant, and police need to rely on other clues such as testimonies. Although assistance has even been sought internationally, both Wager and Vihavainen confirmed that no dependable on-site test exists for this purpose.

Through this meeting, we deepened our understanding of the practical needs in the field. We also discussed what can typically be determined from blood traces at a crime scene. Much like the forensic chemists we consulted earlier in March, Wager and Vihavainen stressed that any new test must be simple, portable, cost-effective, and use only a small portion of the sample to preserve it for other analyses. Additionally, they raised a new important point: the test results must be very straightforward to interpret to minimize the risk of errors at the scene. We were pleased that they found our choice to use a bioluminescent signal promising, and this encouraged us to plan more laboratory analyses to optimize how the signal is detected.

A major topic we explored during the meeting was how new forensic tests are evaluated. Wager explained that completely new tests must undergo extensive validation, often lasting several months, depending on how many samples should be prepared and how many tests should be conducted. For example, with our test the fact whether different aged bloodstains are available for testing would play a big role in the duration of these validations. Alongside this process, a smaller team of quality managers develops essential documentation, which includes explanations of the test principles, quality assurance protocols, and clear guides for crime scene investigators and police officers. Even though our test is nowhere near this step, we felt like it was valuable to understand the whole process a forensic test would undergo before commercial use. Acknowledging this, even in the early stages of our test, helps us document everything in a way that could be useful in the future.

Finally, we discussed the most common reasons why new tests might be rejected. Wager highlighted usability as a critical factor. If a test requires cumbersome expensive equipment or extensive expertise, it is likely to be dismissed, since investigators already have many techniques to employ, and not much excessive time. Wager and Vihavainen expressed confidence that our test would primarily be used in the early stages of investigations to help determine where to focus further forensic efforts. The possibility of widely used in the pretrial investigation luminol component affecting our signal was a concern raised in our interview with Mari Humalajoki (link). Hearing that our test would be used before luminol or other chemical handling was good news. In the future, we still hope to research how other chemiluminescence methods can affect our test's signal. Expert’s concluding encouragement, “If you succeed, this is revolutionary!”, was especially motivating and reinforced the significance of continuing our work.

Finnish Police University College

On August 12th, we met with Chief Inspector Mika Ranki, who serves as the Crime Prevention Expertise Unit at the Finnish Police University College. Ranki has accumulated over 30 years of experience as a police officer and has spent the last 15 years teaching criminal investigation at the Police University College. In the meeting were also Chief Inspector Joni Tonteri, the Superintendent at the Police Dog Training Centre and Sergeant Jarmo Happo, who is a dog handler specializing in cadaver dogs and Sergeant Mika Lemmetti, a teacher at the Police Dog Training Centre. The Police Dog Training Centre falls under the Police University College and supports pre-trial work.

We learned about the structure of education at the Police University College. The bachelor’s degree in police services program is a three-year program that includes an internship at the police department. Furthermore, the scope of studies focuses on patrolling and the timeframe is quite limited. Therefore, the fundamental training focuses on procedures typically conducted by first patrols, such as securing the crime scene, collecting evidence and preserving it using conventional mechanical techniques. These techniques include collecting fingerprints, stain collection and shoeprint analysis with foil and silicone casts. The primary focus of the education is on gathering evidence rather than on its analysis. Bloodstain analysis is not a subject taught at the bachelor’s degree level. It is addressed later in specialized courses.

The specialization courses delve more deeply into the subject matter. These include blue-light searches for biological traces, gunshot residue tests and other targeted methods. These courses are created in collaboration with the forensic lab of the National Investigation Bureau. New tools should adhere to a systematic approach of first piloting the tool and then collecting user feedback. If the tool involves aspects of crime scene practice, it should undergo an audit to eliminate potential error sources before wider implementation.

We received valuable insights from Happo and Lemmetti regarding the use of police dogs, which we had not previously considered a key component in the implementation of our test. Dogs are capable of detecting blood, even old or wiped stains, but they lack the ability to tell the age of the bloodstains. The establishment of a reliable method for distinguishing between fresh and aged bloodstains would represent a significant milestone. As we received yet again the confirmation that typical crime scenes contain bloodstains of different ages and often there are arguments about the relevance of a seemingly old bloodstain. While we had decided to use nanobodies that are not all human specific, the dogs can differentiate samples between humans and animals by scent. Therefore, our test could be complementary to the work of police dog handlers instead of being a competitive method.

We also received the confirmation that even distinguishing between bloodstains under two weeks of age and those which are older helps to efficiently find the relevant bloodstains on the crime scenes. As Ranki noted, “To lose time is to lose the truth.” Results, whether obtained in hours or days, are valuable, even if they are read out in a lab rather than on-site. At the crime scene the focus is on appropriately preserving the samples so an on-site test is not necessary. However, as with any case, time is of the essence. The faster the investigation is conducted, the more likely achieving a successful outcome is. We received again the impression that the timeline of the crime is now reconstructed by connecting a great amount of miscellaneous evidence and just the best guesses, which we had already learned from the interview with Senior Detective Constable Samuli Lehikoinen.

To fulfill the requirements for the test, it is necessary to conduct practical experiments to ascertain how humidity, for example, affects our test. Preserving the DNA in the sample is of the utmost importance. We have already considered this requirement, and as our test does not contain anything destructive and many of the steps of our test are the same with DNA sample collecting. Therefore, we believe that the DNA remains undamaged. In addition, our test should not affect further analysis, for example, tests using luminol (link to interview with Linda Wager and Anu Vihavainen). Due to time constraints for our project, we lack the resources necessary to perform all the required tests needed to determine the margins of error. Thus, we will have to base our assessment of the risks on the available research and interviews with experts.

We also gained insight into the extensive considerations crime scene investigators must take into account. To find the truth, it is essential to demonstrate that no alternative chain of events is plausible as the crime scene investigators search for the material truth compared to the procedural truth the court looks for. In addition, we wanted clarification on the terms “objective evidence” and “subjective evidence” that we had previously utilized in past interviews with professionals. We observed there was a difference in interpretation in our understanding of the definition of these terms to the professionals involved. We learned that when referring to evidence that is obtained technically, the terms “objective evidence” or more commonly “technical evidence” are used. However, the term “subjective evidence” proved to be more confusing, and we were advised to use the term “witness testimonies” instead, as it clearly conveys our intended meaning.

We also learned about the possible future steps in the development of our test. Once the test is ready, we should reach out to the National Police Board to discuss the further development of our test, as the Police University College main focus is in teaching the most common methods. We also received a useful tip regarding presenting our project. In order to make the subject more interesting and easier to understand to a wider audience, it would be beneficial to link it to a practical example while presenting our project.

Law

Doctor Markku Fredman

On June 4th, we met with Doctor Markku Fredman, a lawyer, working at the University of Helsinki as professor of practice. Drawing on his expertise as a practicing lawyer and specialist in procedural law, pretrial investigation and coercive measures, he walked us through how our test might fare as evidence in court. We received clear feedback on legal fit, evidence rules and ethical guardrails.

We learned that the real strength of our test lies in guiding crime scene investigations rather than serving as evidence in court. By distinguishing recent bloodstains from old ones, our method can point investigators to the most critical areas, saving both time and resources at the scene.

Along the way, we received plenty of new information about the Finnish legal system. Courts in Finland use free appraisal of evidence: though any lawfully obtained data can be presented, its weight is determined by judges and expert witnesses. To earn that significance, our test must build a solid reliability record. Moreover, the test would not need to pinpoint the blood stains’ age down to the hour, since other evidence will also be presented in court. Even a broad timing window adds value to our test if we back it with transparency, repeatability, and validation data. In addition, the Finnish justice system heavily trusts subjective evidence, which isn’t reliable given the unreliable nature of human memory. Hence, there is a need for a test that could provide objective evidence.

We also learned about the Coercive Measures Act, which allows the use of invasive techniques, like taking a sample from a suspect for DNA sequencing, only in cases where the suspected offense carries at least a six-month sentence. Because our test works on on-scene material rather than on direct human samples, it skirts the deepest privacy concerns. However, clear documentation on how material is collected and handled is still needed. The following table illustrates how privacy would be assured.

Step	Privacy safeguards
Sample collection	• Wearing gloves and changing them between samples • Using sterile, single-use swabs • Recording the date and collector’s name • Photographing sample location
Drying and packaging	• Air-drying swabs • Sealing evidence bags with initials and date • Logging evidence with unique sample ID (no personal identifiers) • Using a chain of custody form
Transport and storage	• Recording each hand-off in transport log • Transporting via secured courier in a locked evidence box
Analysis and disposal	• Test performed only by crime scene with the appropriate training • Documenting disposal method and date

Table 1. Privacy safeguards used with our test, adapted from [2]. ‌

As our test is non-profiling, it offers a significant advantage by eliminating many ethical issues that could be related to profiling tests. There is also no legislation that prevents crime scene investigators from using it in “just in case” scenarios as it does not classify an invasive technique. We also discussed the potential for misuse, though it seemed unlikely. The only possible scenario that was presented to us was that someone who knows about the test, would return to the crime scene and scatter other people’s blood to mislead investigators, yet even that is highly unlikely.

Initially, we had envisioned that our test would be used in court, however, after the meeting, we realized that it could only be used for alibi demonstration in court. Furthermore, the evidentiary value of our test would diminish, once the police obtains more precise evidence, such as confessions. Nevertheless, after learning that it might be more useful in the pretrial investigation our approach to pitching our test shifted. Our test could significantly cut down time off crime scene searches as the investigators could focus straight away on the relevant bloodstains. We realized that is how we should present our test.

Given that, we realized that our primary audience is actually not lawyers and judges, but rather crime scene investigators. Forging connections with busy, confidentiality-bound investigators became our top priority. Overall, our conversation with Markku Fredman sharpened our understanding of the Finnish justice system and showed us that the greatest impact of our test will be at the scene, not in the courtroom.

Doctoral Researcher Esko Yli-Hemminki

On June 6th, we met with Esko Yli-Hemminki, who is a Doctoral Researcher at the University of Helsinki. He specialises in criminal law, legal theory, and the philosophy of punishment. We wanted to better understand the role our test could play in criminal investigations and the Finnish justice system. Our conversation with Yli-Hemminki reinforced our understanding about how our test would not serve as sufficient evidence in court. Instead, it would act as a supporting tool during preliminary investigations, helping investigators make faster and more informed decisions at the crime scene. This shifted our focus away from the courtroom towards the practical utility of our test in real-time investigative work.

Yli-Hemminki provided valuable insight into the structure of the Finnish justice system, particularly the role of objective and subjective evidence. While objective evidence has a higher display value in court, it is not sufficient on its own for a conviction. Subjective evidence plays a critical role in interpreting objective evidence. In addition, even objective evidence needs to be interpreted. Thus calling evidence objective or subjective is misleading. He introduced us to the concept of the conviction threshold, explaining that the court must be left with no reasonable doubt that a crime did not occur in order to deliver a guilty verdict.

Another important point raised during our discussion was that police officers and investigators must clearly understand how the test works, including its limitations and scope. In response, our next steps include contacting law enforcement professionals to gather their perspectives on our test’s usefulness and exploring how it could be improved to better meet their needs.

Yli-Hemminki also highlighted a key ethical concern: a high margin of error could lead to wrongly directing suspicion toward innocent individuals. To acknowledge this, we talked about the issue with multiple stakeholders in the fields of forensic science, crime scene investigation, and ethics to examine this issue more deeply and guide us in minimizing such risks.

Initially, we believed that the value of our test would lie in reducing the backlog of samples sent to crime labs, thereby speeding up investigations and potentially even saving laboratory materials. However, during our conversation, we learned that serious crimes like homicides are most likely prioritized in laboratories and might be processed quickly. This new insight led us to revise our understanding of the value of our test: its greatest strength lies in providing immediate, on-site information that can help investigators make faster, more accurate decisions at the crime scene.

Overall, this meeting helped us reframe the role of our test within the broader justice system and clarified both its potential benefits and the responsibilities we must consider as developers.

Professor Kimmo Nuotio

On June 11th, we met with Professor Kimmo Nuotio, who is a Professor of Criminal Law at the University of Helsinki. He also serves as Director of the Institute of Criminology and Legal Policy and chairs the Research Council of Finland.

One of our initial concerns was whether our test could lead to an overemphasis on technology in criminal investigations, particularly if the situational context of a crime scene is not fully understood. Nuotio supported this concern by sharing an example from Lake Bodom murders, which is a well known Finnish homicide case. During a later investigation into this case, technological findings were overly interpreted due to a lack of holistic analysis. While he reminded us that, legally, convictions in Finland cannot be made solely on technical evidence, the case highlighted how early-stage technology could unintentionally guide investigations in a particular direction.

This emphasised the importance of documenting our test’s scope and clearly communicating its limits. Nuotio also stressed that, even in a competitive setting like iGEM, we must not overstate the capabilities of our technology. Transparency about the test’s current stage of development and its limitations is essential to prevent misunderstandings or misapplication. This is something that we have kept in mind throughout the whole process.

Nuotio brought up the point that, because our test is a tool for preliminary investigation, it avoids many of the ethical and legal complications associated with evidentiary or final forensic analyses. He felt that the risk of misuse of our test is minimal. This led us to conclude that our main ethical responsibility lies in ensuring the test is accurate, transparent, and that its principle of use is well-documented to avoid any misunderstandings.

We also discussed preventive policing, where data and tools are used to anticipate and prevent crimes. While our test is not designed for this purpose, Nuotio raised the point that technologies used in such contexts can reinforce structural bias or discrimination, for example, by perpetuating a focus on certain neighborhoods. Although we learned that this should not be a concern in the European Union as there are regulations regarding the problem, this is still something we will keep in mind throughout the development of our project.

Finally, we learned about the principle of “beyond a reasonable doubt”. This principle reflects a strong ethical commitment to avoiding wrongful convictions: it's considered better that ten guilty individuals go free than for one innocent person to be wrongly convicted. This concept made a strong impression on us and is something that we implemented in the consideration of how to communicate about our test reliably.

Doctoral Researcher Jani Hannonen

On the 12th of June, we met Jani Hannonen, a Doctoral Researcher at the University of Turku specialising in criminal law, human trafficking, recorded interviews and Barnahus. When we first contacted Hannonen, we had little to no knowledge about the legal process in Finland. We were also unsure of where exactly our test would fit in a criminal procedure.

His expertise on recorded interviews was especially exciting to us. Human memory is malleable, and the legal process can take years, so recording interviews early after the crime can help contain important information that could otherwise be lost or altered at the time of the trial. Recording interviews can also be useful when children are involved. Why we were excited about this expertise of his, however, was because this told us that he would know about the characteristics of good evidence and also about preserving evidence.

We learned that good evidence isn’t changed by the passage of time. Human memory, for example, is quite unreliable so using recorded interviews can be beneficial. However, the justice system values testimonies given at the trial more than interviews recorded beforehand even though it’s less reliable.

Free evaluation of evidence means that everything can be used as evidence as long as it has been acquired legally. This is policy in Finland, and it is also common in many countries. Before meeting with Hannonen and other people specialising in law, we had thought that our test would need to meet some requirements to be used as evidence. Thus, learning about free evaluation of evidence was surprising to us.

Hannonen told us that free evaluation of evidence means that there isn’t, for example, any precision or accuracy requirements our test needs to meet to be used as evidence. However, being more precise is always better but he reassured us that our test doesn’t need to give an absolute time. If our test is really expensive and unreliable though, it probably wouldn’t be used at all. Hannonen also helped us confirm that the greatest value of our test is in pretrial investigation and verifying alibis.

We also learned about the differences between subjective and objective evidence. Hannonen sees objective and subjective evidence as intertwined. While objective evidence gives us facts, we still need subjective evidence to give us the context. In addition, objective evidence still needs to be interpreted by an expert, making it not purely objective by nature. Furthermore, subjective evidence is, in his opinion, overvalued but often it is all that is available.

We had also been wondering if using our test at the crime scene had any legal concerns. Hannonen told us that our test wouldn’t violate protection of privacy as the sample for our test would be acquired from the crime scene and not from a person. Our test wouldn’t also give any information about the donor of the bloodstain. Using bloodstains to find information about the donor of the bloodstain is, however, acceptable if there is a reason for it.

Once we had confirmed that our test had no legal concerns, we moved to discuss ethical issues. Hannonen saw no ethical issues with our test. He saw no potential of misuse because our test doesn’t give any sensitive information about the donor. Human rights are well established in Finland, so he doesn’t see giving more tools, especially non-profiling ones, to the police as a major concern.

Hannonen was also excited to help us with recruiting the next ABOA team. As a team we have been talking about wanting the next ABOA team to be more multidisciplinary. This year’s team has quite a homogenous academic background, which has its own benefits but also downfalls. While iGEM is at its core a synthetic biology competition, we have learned that only knowing about synthetic biology won’t get us far. We need people who know about communication and graphic design, for example, to truly be successful. Thus, Hannonen’s suggestion to spread the word of iGEM to law students was an exciting one.

Synthetic Biology

Doctor Aapo Knuutila and Doctor Kirsti Raiko

On March 19th, we met Aapo Knuutila (PhD) and Kirsti Raiko (PhD). The goal of the meeting was to guide our research into a feasible path for a solution. At the beginning of the meeting, we presented the concept of iGEM, our findings on our project topic so far, and reflection on possible directions to developing our solution.

We presented multiple possible biomarkers and our estimation of their viability for a bloodstain age determining test. However, we had identified a challenge—the number of studies on bloodstain degradation is very limited. The professionals advised that in this case, our main focus should be biomarker discovery. Out of the proposed biomarkers, human serum albumin, cortisol, and melatonin stood out as the most promising ones. Raiko also suggested that instead of measuring the change of an intrinsic biomarker, we could measure something that transfers from the environment to the bloodstain.

For the design, we had entertained the ideas of an immunological and a receptor-based approach. We learned that while a receptor-based assay is possible, we would need to begin the design by identifying a signalling pathway for the analyte and searching whether there is something to measure in the signalling pathway. For an immunoassay, we would have many possible methods of detection and the production of the parts would probably be easier. Additionally, we gained some inspiration from various models of immunoassays.

We had noted that not knowing the sample's original quantity might raise an issue. The professionals affirmed that we might not have to solve the original volume. This could also be beneficial if the sample size is very limited. We also learned that measuring multiple analytes with the same test could be possible. For example, in the case of measuring circadian rhythm hormones—such as melatonin and cortisol—we could use a bispecific assay system with two different labels. The assay could also be applicable to a lateral flow test that would have two test lines. However, for hormones, immunological detection would be difficult due to their small size.

At this point, we had already considered objectives for the real world application. The two main points were that the test should be on-site and it should give a quantitative signal. We wanted insight from the professionals into what kind of limitations or challenges these objectives presented for the assay and the product design. We learned that for example, for the most common type of an easy-to-use on-site test—a lateral flow test—it is possible to get a fluorescence signal and portable readers for this already exist. However, the professionals stated that practicalities related to, for example on-site application, can be adapted and improved after the basic principle of the assay has been proven to work. At first, the emphasis should be to think about what is the synthetic biology part of our project and what is the novelty appeal. As for the process, we should start with focusing on the biomarker, then think about the detection and only last think about the reporter and signal.

We also discussed the practical testing of our assay in the meeting. Initially, we had planned to use bovine blood as the matrix for our testing due to accessibility and safety reasons. However, we learned that while the albumin proteins have similar structures, detectors for human serum albumin might not recognize bovine epitopes. Thus, we were advised to use human blood and educated on the safety and regulatory aspects of working with human samples, such as the Declaration of Helsinki.

Additionally, Knuutila supported our team across multiple technical, ethical, and logistical challenges. We have contacted Knuutila with questions regarding blood-handling safety, necessary vaccinations for working with blood, and Check-In form for human blood use. He strongly recommended hepatitis B vaccinations for people working with blood, and helped us establish guidelines for working with blood. Knuutila confirmed that the blood would be vacuum-drawn into anonymized EDTA tubes. With Knuutila’s support, we were able to acquire human blood and HSA from the Biotechnology Department of the University of Turku.

Knuutila’s and Raiko’s combined support—ranging from practical materials and regulatory guidance to technical troubleshooting—along with Knuutila’s logistical aid, which made our wet lab work possible, had a significant impact on the success of our project.

Professor Urpo Lamminmäki

We have had 4 meetings in total with Urpo Lamminmäki, who is a professor of Biotechnology specializing in antibody engineering, bioaffinity assays, and in vitro diagnostics. On top of these meetings, Lamminmäki has offered help in acquiring necessary equipment and reagents throughout the project.

April 11th 2025
We had our first meeting with Lamminmäki on the 11th of April, when we were still quite early in the project. The goal of the meeting was to get insight on the biomarker, the detector molecule, and the actual assay method for determining bloodstain age.

Lamminmäki commented that a good biomarker for our envisioned on-site test would have standard concentrations in blood and its concentration would decrease as bloodstains age. Lamminmäki theorized that in dried bloodstains the degradation of proteins is mostly caused by oxidation and other chemical reactions. When the bloodstain is still wet, proteolytic activity is the key factor causing protein degradation. Proteolytic degradation is usually much more efficient than degradation by oxidation, which is why we should verify the extent of the effects caused by oxidation to our biomarker protein and how fast they occur.

We had done literature research on human serum albumin (HSA) and found that common binding molecules for HSA are usually antibodies, antibody fragments or virulence domains of bacterial proteins. While we were unfamiliar with antibody or antibody fragment production, the thought of using potential virulence factors also seemed unappealing and a potential safety hazard. Lamminmäki commented that virulence factors such as albumin binding domains (ABDs) would most likely be easy to produce in bacteria as they are of bacterial origin. He did not see using ABDs as a safety issue, as these proteins are commonly used in industry, medicine and antibody purification for example. For addressing safety concerns, Lamminmäki proposed using a laboratory strain as it would not be pathogenic.

Despite antibodies being more difficult to produce than ABDs in bacterial hosts, Lamminmäki did not see any real issue with producing antibody fragments in E. coli. He proposed using a binding molecule, whose epitope is prone to degradation so after the protein has degraded we would not be able to detect the protein any longer. Thus, we shifted our focus towards looking more into the epitope stability of the known binding molecules rather than deciding which type of binding molecule we should use.

Looking at databases such as PDB for X-ray crystallography structures of our chosen biomarker crystallized with a binding molecule was recommended by Lamminmäki. For evaluating the stability of the epitope, it was suggested to look at the amino acids of the epitope. Methionines and tryptophans are especially oxidation prone amino acids.

Lamminmäki also brought up the idea of using an enzyme fragment complementation assay (EFCA). The assay could be performed homogeneously, meaning without washes, which is a clear advantage for an on-site test. Lamminmäki emphasized that we should pay attention to the choice of our split enzyme as the signal it produces should either be visually interpretable or able to be read with a simple device on-site. For HSA, a highly sensitive assay is not needed, rather attention should instead be paid to find the correct dilution ratio as HSA concentration in blood is quite high.

Prior to our meeting, we had had concerns regarding having to know the original volume of the bloodstain for the assay to be accurate. Lamminmäki addressed this concern by proposing doing two measurements instead of one: one measurement, where the binding molecules bind to stable epitopes, and another where the binding molecules bind epitopes that degrade over time. Thus, we set a goal to find at least 3 epitopes on our biomarker: two stable ones and one unstable one.

The first meeting with Lamminmäki was an exciting one and laid a foundation for our whole on-site test, VeriFied. Based on our own research on literature and the discussion with Lamminmäki, the basic premise had now been laid out: VeriFied would be an on-site EFCA-based test that uses nanobodies to bind a biomarker whose concentration decreases as bloodstains are exposed to oxidative environments during the aging process.

May 9th 2025
The goal of our second meeting was to get an expert's opinion on linker design, using tags for protein purification, using luciferase as a reporter enzyme, what kind of variations of the fusion proteins should we focus on, periplasmic expression, and bacterial strain. At this point in our project, we had made the decision of using HSA as our biomarker and EFCA as our assay method.

We had found a nanobody library article by Shen et al [3]. Using crosslink-mass spectrometry data and nanobody sequences provided by the article, we modeled the epitopes of the nanobodies to evaluate the stability of the epitopes. However, we were still quite unsure of the linker connecting the nanobodies and enzyme fragments and wanted to get Lamminmäki’s opinion on it. Lamminmäki recommended making the linker a bit too long rather than risk making it too short. He noted that too long linkers decrease the probability of binding but a too short linker would inhibit the binding as envisioned completely. GGGGS linker was agreed to be a good flexible linker sequence. The optimal linker placement would be from the nanobody C-terminus to the enzyme N-terminus as it would not hinder enzyme fragment binding.

Attaching tags to the nanobody C-terminus would likely not be an issue according to Lamminmäki. In the enzyme fragment’s split site, it could however hinder the binding of the fragments. It was proposed to use Hisx8 as a tag for purification. Hisx6 would be sufficient as well.

Although we had come across the use of luciferases in EFCAs, they did not fit the original idea of using an enzyme that could produce a visually interpretable signal. Fortunately Aapo Knuutila and Kirsti Raiko had informed us of portable luminometers so luciferases could still function as the reporters for an on-site test. Lamminmäki brought split NanoLuc into our attention as a luciferase that is quite commonly used in EFCAs.

For fusion protein variations, we had thought of either doing fusion proteins with varying nanobodies or varying linker lengths as these seemed like the potential factors to optimize. Lamminmäki encouraged us to focus the variations on nanobodies rather than the linker. We also got a suggestion to design a fusion protein with whole NanoLuc instead of just a fragment of it. This kind of protein could be used to test the binding of the nanobodies in a heterogeneous assay without the additional variable of the EFCA.

Making the choice between periplasmic and cytoplasmic expression was discussed in the meeting as well. Periplasmic expression would ease the nanobody expression as the oxidative conditions of the periplasm enhance the formation of disulfide bridges. However, with periplasmic expression, the expression levels are lower than with cytoplasmic expression. In the end, we decided to proceed with periplasmic expression, because if the nanobodies would not fold correctly in the cytoplasm, refolding them, especially because they are fusion proteins, could prove immensely difficult.

Between the first two meetings with Lamminmäki, we had also had time to research the expression strain more. BL21(DE3) seemed promising to us. Lamminmäki reminded us that if we wished to do molecular biology, BL21(DE3) would not be most suited for our purposes as it still has nucleases.

The second meeting was spent refining our assay design. Many important factors about bringing VeriFied to life such as deciding to use periplasmic expression, His-tag, and NanoLuc were adapted to our design based on this meeting.

May 22nd 2025
We invited Lamminmäki to assist us for the third time when we wanted our DNA construct designs proofread. Our PI Pauli Kallio also attended the meeting. During the meeting, all ten of our constructs were thoroughly inspected and commented on as well as our plan for producing and purifying our fusion proteins. You can find our DNA construct designs here and more about later DNA construct redesigning efforts here.

The chosen plasmid backbone pET-IDT C His includes Hisx6, and Lamminmäki proposed to add Hisx2 to the end of the insert, so the fusion proteins would have Hisx8. We still had concerns of the His-tag affecting the binding of the enzyme fragments, so Lamminmäki proposed looking into studies where NanoLuc fragments with His-tag were used. As a safety measure, we got a proposition to design a protease site before the His-tag so it could be removed after purification if any issues would arise.

Suggestion to use XL-1 Blue strain for potential molecular cloning was also brought up. As we had decided to order our DNA constructs in expression plasmids, we did not end up using the XL-1 Blue strain at all. All of our lab work was performed with the BL21(DE3)pLysS strain.

Overall, the meeting served as a big confidence boost for us. Lamminmäki and our PI Kallio made sure we had reasoned every choice about our constructs, and they were impressed with the explanations we provided them with. For us, the praise we received from them made us feel like all the hard work we had done working on the design finally seemed to bear fruit. We placed the order for our plasmids pleased with all the skills we got to learn while designing them.

August 27th 2025
After we had spent the whole summer in the lab, it was time to get some assistance from Lamminmäki once again for evaluating the results we had gotten. We also wanted comments on the presentation of our end product, VeriFied.

Spending the whole summer in the lab had left us with quite a lot of data, some of which we were not completely sure possessed any true significance. Lamminmäki encouraged us to emphasize the fact that we were able to express our fusion proteins, that the test concept works, and that there is a trend between luminescence signal and bloodstain age.

Many ideas for future experiments and optimization were proposed as well. Lamminmäki recommended that for potential future experiments it could be wise to just lyse the cells instead of doing the periplasmic extraction. Lysis could however increase the risk of our fusion proteins degrading due to protease activity. Cytoplasmic expression could also be tried to achieve higher expression levels. Optimization of the expression otherwise should also be continued.

The fusion proteins need to be purified if we want to commercialize the method. Although we got preliminary results indicating some success with our test concept, more measurements are needed to validate the initial data we managed to gather. Concentrations of all test components and the actual measurement protocol should be optimized as well.

There are still many variables we need to take into account before VeriFied can be brought to crime scenes. Biological replicates should be tested as well as bloodstain samples that have been aged differently, outdoors for example. Environmental factors have been a key reason for many crime scene investigation methods not being adopted for use. By using western blot, it would be possible to verify that the nanobodies do not bind to any other protein besides HSA. In our interview with the staff from PolAmk, a wish for human specific nanobodies was expressed as well. Optimization of the nanobodies used definitely deserves a spot on the future endeavours list. You can read more about our ideas forfuture experiments here .

We had been wondering whether our test could pose any safety concerns to the end users as it contains the nanobody components that bind HSA, a protein found in human blood. Lamminmäki did not see this aspect of our test as a safety risk.

It was especially rewarding getting to present our idea for the end product to a person such as Lamminmäki who had been with us from almost the very beginning of our project. You can read more about how we envisioned the end product and the associated protocol here.

Professor Olli Pentikäinen and Doctoral Researcher Paola Moyano-Gómez

We contacted Professor Olli Pentikäinen and arranged a meeting on the 25th of April to discuss ways to evaluate the effects of oxidation on epitope stability on the surface of human serum albumin (HSA). At this point in our research, we had found an article by Shen et al. detailing a large library of high affinity nanobodies that bind to different epitopes on HSA [3]. We needed help in finding epitopes located on areas likely or unlikely to have structural changes due to oxidation. Since no one on our team had significant experience in 3D modeling protein-protein complexes or running docking simulations, we additionally asked for help with learning the techniques.

Before the meeting, we had done some analysis of areas on the surface of HSA that were likely to undergo structural changes due to oxidation. Our previous analysis was largely based on finding oxidatively reactive amino acid residues such as methionines, tryptophans, and free cysteines. Professor Pentikäinen pointed out that in addition to sequence analysis, structural interpretation should be taken into account since secondary and tertiary structures can cause certain residues to be more sheltered, reducing their reactivity. Still, he emphasized that finding individual residues significantly affected by oxidation would most likely be the best way to find an unstable epitope since they are likely to cause local structural changes due to oxidation. Cys34, Met87, and Met329 were raised as particularly promising, oxidation prone residues.

After asking about computational methods to assess oxidation prone areas and structural changes caused by oxidation, Professor Pentikäinen recommended studying literature for previous research done on the subject since computational predictions can be unreliable. He explained that most tools used for predicting structural changes in proteins are developed for evaluating the effects of point mutations, meaning they would likely be unsuitable for predicting structural changes caused by oxidation.

Professor Pentikäinen additionally pointed out that the slightly sheltered location of Cys34 between domains I and III of HSA could be beneficial for our test since it lowers its oxidative reactivity. If it was fully exposed on the surface of HSA, it would likely react very fast when exposed to oxygen, meaning our test could only predict the age of very recent bloodstains. Our test having a wider time window would allow it to be used in a larger variety of crime scenes. He also mentioned that the total size of the blood stain is a variable that could affect oxidation since it affects the surface area of blood directly exposed to air. The test would have to be validated for this variable before use.

Most of the nanobodies we were considering using were from a high affinity HSA binding nanobody library by Shen et al. [3]. They used crosslink mass spectrometry and size exclusion chromatography to study the epitopes of the nanobodies, which leaves the epitopes slightly ambiguous, but Professor Pentikäinen assured us this would most likely be accurate enough for our purposes. Since the structures of the full nanobody-HSA complexes were not released in the paper, he suggested using homology modeling to predict the shape of the nanobody, followed by protein-protein docking using software such as HADDOCK with the crosslink data used as constraints to predict the epitopes. Of course if we managed to find nanobodies with experimentally solved nanobody-HSA complex structures, those would be preferable since the exact epitope and orientation are known.

Next, we asked about what we should take into account when designing the linker for our fusion proteins. Professor Pentikäinen explained to us that usually linkers are designed to be longer than needed to make sure the fusion protein works properly, and the linker length is later optimized. Longer linkers are more vulnerable to proteolysis by proteases and reduce the efficiency of the enzyme fragment complementation, but if the linker is too short, the test won’t work at all due to steric hindrance. Deciding which terminus of the protein to attach the linker to is also a crucial step since it can affect things such as nanobody binding and enzyme fragment complementation. Since we do not have experimentally validated structures for all our nanobody-HSA complexes, there is uncertainty of the exact orientation and epitope of the nanobody. This means we should design our linkers to be longer than necessary to account for this uncertainty.

At the end of the meeting, Professor Pentikäinen mentioned the possibility of doing a mixed solvent molecular dynamics simulation for finding likely binding sites on the surface of HSA but we deemed the method too complex and time consuming for our project. This idea could, however, be pursued by a future team to test our hypothesis further.

After the meeting, Professor Pentikäinen invited us to the Institute of Biomedicine to help with 3D modeling various nanobody-HSA complexes with the help of Doctoral Researcher Paola Moyano-Gómez using BODIL. The goal was to superimpose all of the nanobody-HSA complexes found on PDB and the ones we modeled based on the article by Shen et al. on top of each other to find the most promising ones, and ones that do not compete for the same binding sites. Additionally, we were able to use the institute's 3D glasses and projection equipment to help us visualize the structures better.

During the modeling, we found a nanobody from the article by Shen et al. (Nb13) that had an epitope on the opposite side of domain I from Cys34, but there were alpha helixes beginning from Cys34 leading to the binding site as seen in Figure 1. This means that the oxidation of Cys34 might cause conformational changes to the alpha helix, which could be a significant enough structural change to impact the affinity of Nb13.




Figure 1. Nb13 epitope in relation to Cys34


Prediction of Nb13 binding site based on docking run using cross-link mass spectrometry data by Shen et al. Cyan: Nb13, green: HSA, magenta: Cys34, beige: structurally interesting alpha helixes.

The meeting with Professor Pentikäinen and hands-on modeling with Moyano-Gómez helped guide our choice of nanobodies and gave us important insights into our fusion protein design. Choosing nanobodies that are able to bind to HSA at the same time without steric hindrance and designing the optimal linkers for our purposes are critical steps to ensure the success of our project.

The Faculty of Science and Engineering

On 6th of May, we met with Tiina Salminen, Mia Åstrand, and Tomi Airenne from Åbo Åkademi University Faculty of Science and Engineering, unit of Structural Bioinformatics Laboratory. We initially contacted Tiina Salminen, a Professor of Biochemistry and Cell Biology, whose research focuses on 3D structures of proteins and how the structure affects protein function. Salminen suggested inviting Mia Åstrand, a University Lecturer in Cell Biology, and Tomi Airenne, a Project Researcher in Biochemistry and Cell Biology, to the meeting.

During our literature research, we noticed a lack of research done on oxidation mediated structural changes of HSA. The purpose of the meeting was to get validation of our design hypothesis and guidance on tools and methods, both digital and physical, for evaluating how oxidation affects human serum albumin (HSA) stability. We presented our literature research findings on the structural changes of HSA caused by oxidation. We emphasized the role of Cys34 in HSA oxidation and showed our prior modelling work. Our work received validation from the experts. They also informed us of the possibility of studying Cys34 oxidation computationally, but we ended up not pursuing that in favor of time.

During literature research, we came across the role of fatty acids in HSA oxidation. Fatty acids can also oxidise, which is why much of the research is done with defatted HSA. In addition, binding of fatty acids causes conformational changes that make HSA more prone to oxidation. We were also wondering if the binding of fatty acids could hinder nanobody binding. As fatty acids might affect our wet lab experiments, we got a recommendation to set a standard for our test regarding fatty acids. Additionally, we got a proposition to make different linker variants. We ended up not pursuing this, because Urpo Lamminmäki suggested focusing the variants on the nanobodies.

Salminen told us about Prometheus Panta, a device that can give insight into protein stability. Airenne, the person responsible for Prometheus Panta at Åbo Akademi, told us more about the technical requirements for performing protein stability measurements on Prometheus Panta. After reviewing the sequence and crystallographic structure of HSA, he confirmed that HSA was suitable for analysis using Prometheus Panta.

Prometheus Panta provides a thermal denaturation curve as a result. It can also be used for measuring sample purity. Airenne proposed that we would test two different HSA samples: one in which Cys34 is oxidised and another where Cys34 has not been chemically treated. Thus, we would be able to see whether Cys34 affects HSA stability.

On 11th of June, we met with Airenne again to discuss the technical details about performing our experiments on Prometheus Panta. In addition to giving us the permission to use Prometheus Panta, we were offered support with data analysis. On 20th of August, we performed the analysis with Prometheus Panta with the help of Mikko Huhtala, MSc of Biochemistry and Cell Biology. He also assisted us with data analysis.

During the research phase of our project, we noticed how limited research on HSA oxidation is, especially outside of the medical field. We set a goal to do our own empirical research on HSA stability. This motivated us to carry out empirical studies ourselves. The opportunity to use Prometheus Panta became a crucial element in achieving this goal.

Doctor Jarmo Niemi

In late June 2025, our team contacted Professor Mikko Metsä-Ketelä regarding challenges in separating human serum albumin (HSA) from hemoglobin in reconstituted blood samples. Metsä-Ketelä had previously lectured on protein precipitation techniques for X-ray crystallography, which made him a natural first point of contact when we encountered difficulties with protein-level separation. The inquiry included our previous Bradford and hemoglobin measurements but lacked spectrophotometric quantification of HSA, which the team was not yet aware was feasible.

Following the exchange, Metsä-Ketelä forwarded a suggestion from Doctor Niemi: to explore bromocresol green (BCG) as a method for direct spectrophotometric quantification of HSA. Along with this recommendation, Niemi shared a selection of helpful scientific reviews covering albumin measurement techniques, protein precipitation methods, and fractionation strategies relevant to our work.

Inspired by this, our team requested the BCG reagent from the Department of Chemistry. Tiina Buss, a lab technician of the Department of Chemistry, provided our team a 4 g vial of the dry reagent.

When we were looking for facilities to incubate E. coli at 16 °C, Niemi inquired about the available incubation infrastructure within his unit on our behalf. Unfortunately there were no suitable incubators, but luckily after talking to Sami Oksanen and Eeva-Christine Brockmann we realized that 26 °C might work equally well.

Tommi Riihinen

On the 21st of May, we had a meeting with Tommi Riihinen, who was the ABOA 2024 team co-leader. Riihinen is also the Chairperson of Aboa Turku ry and a bioengineering student. The goal of the meeting was to present our plasmids and project design and get feedback on them.

We presented our 10 plasmids to Riihinen 7 of which are used in our on-site test, while 3 are controls. The 7 plasmids give us 9 potential nanobody pairs for our on-site test and the 3 controls aid wet lab troubleshooting. Riihinen suggested the option of ordering all the parts in one plasmid and using PCR primers to make even more variations. Nonetheless, our aim of ordering the plasmids expression ready made sense to him in favour of time.

Before the meeting, we had planned ordering our expression ready plasmids in a pET-21b(+) backbone. However, Riihinen encouraged us to order from IDT, an iGEM sponsor. IDT offered iGEMers free shipping, which is a significant advantage due to our financial difficulties. IDT offered a plasmid called pET-IDT C His, which turned out to be mostly similar to pET-21b(+). Because of Riihinen’s suggestion, we decided to order our expression ready plasmids from IDT, which meant having to edit our initial plasmid designs.

Additionally, we presented the proteins the genes in the plasmids code for. Riihinen commented that optimizing the linker length would enhance our test’s performance, but optimization should be done at later stages. We told him that we had checked that the linker is always long enough, which Riihinen told us to be sufficient at the moment. Riihinen also noted that using AlphaFold to model nanobodies instead of homology modelling, which we had done, could be more accurate. However, he thought it’s best to proceed to the wet lab than to continue modeling as we already had 7 good nanobody candidates.

Riihinen was also interested in our choice of NanoLuc for a split enzyme. NanoLuc is small, precise and the bioluminescence signal is easy to measure on-site. We had considered using beta-lactamase, which gives a colorimetric signal, but it proved problematic. As our sample matrix is blood, we were worried about the background signal being too strong in a beta-lactamase assay. Riihinen suggested looking into fluorescent proteins as a backup plan, because they might be more favorable financially as they don’t require a substrate.

We also presented our initial thoughts for wet lab such as our plans to do experiments with blood to study HSA oxidation. Riihinen suggested the option of trying to acquire more blood in autumn for conducting more experiments, which we didn’t end up pursuing in the favor of time and other resources. Some diseases cause HSA oxidation, which Riihinen reminded us of. As we don’t have the resources to test the effects of these diseases on our test, we have done literature research. He recommended us to estimate the amount of people with such diseases and to be open about the margin of error these diseases cause.

On week 25, we consulted Riihinen again, because IDT cancelled 8 of our 10 plasmids because they didn’t pass the NGS test. Riihinen helped us to figure out why the plasmids got cancelled and to redesign them. The linker sequence is GC% rich, which is a potential reason for the synthetization failure. Some nanobodies also have a GC% rich area right before the linker sequence which amplified the problem. There were also areas where codons repeated ≥3 times, which increases genetic instability and the probability of polymerase sliding off. Together with Riihinen, we designed a better linker nucleotide sequence and codon optimized the entire insert sequence using codon optimization tools provided by IDT and Twist Bioscience.

After redesigning the constructs, we decided to order the optimized constructs from both IDT and Twist Bioscience. We received NanoChuck and Nb126-SmBit in pET-IDT C His from IDT from the first order and Nb29-Nanoluc in pET-IDT C His from the second order. All the rest ended up being cancelled. From Twist Bioscience we received the rest of our 8 plasmids in pET-21(+).

Associate Professor Saara Wittfooth

Associate Professor Saara Wittfooth is a researcher and teacher at the University of Turku, specializing in clinical biochemistry and biotechnology. Her expertise includes modern analytical methods, protein chemistry, and diagnostic assay development. She teaches in the Faculty of Technology and has extensive knowledge about blood based biomarkers and immunological assay development. Throughout the ABOA 2025 project, she supported us as an external expert, offering critical insight into protein degradation, matrix effects, and purification strategies.

February 2025
Towards the end of February, we had a brief discussion with Wittfooth regarding potential bloodstain age determination strategies. During the conversation, Wittfooth raised the issue of matrix effects, pointing out that purified proteins age differently than those embedded in whole blood. This led to the conclusion that authentic degradation processes could not be reliably simulated or detected outside the blood matrix. The idea ultimately contributed to the decision to work with dried and rehydrated whole bloodstains instead of purified albumin solutions.

May 2025
In May, we sent Wittfooth a detailed email describing a proposed proof-of-concept assay. The aim was to evaluate whether different nanobodies would bind differently to oxidized forms of human serum albumin (HSA), depending on the protein’s age. The setup was intentionally simple and designed to be cost-effective, involving GFP-tagged nanobodies, biotinylated HSA immobilized on streptavidin-coated 96-well plates, and direct fluorescence detection.

In her response she pointed out several technical and conceptual limitations of the proposed strategy. She noted that many oxidative modifications are unspecific. She emphasized that the specific cause of the lowered binding affinity is hard to measure directly. At the same time, she acknowledged that the general idea had merit and suggested that the later design should also have a method to detect albumin in general, regardless of age.

Importantly, she also predicted that full hemolysis was likely to occur when working with dried and rehydrated bloodstains. While this point did not receive immediate attention from the team at the time, it later proved crucial. This realization, which only fully registered with us in early June, directly motivated the development of purification strategies—and ultimately led to the brainstorming session with Wittfooth in late June.

The feedback helped us reassess the analytical feasibility of the proposed test and underscored the importance of having fallback strategies if the full enzyme fragment complementation system failed. Although the proof-of-concept assay was never implemented, the questions it raised—and the overlooked warning about hemolysis—shaped our experimental planning in the following weeks.


June 2025
In late June, we contacted Wittfooth to request a focused brainstorming session concerning hemoglobin removal from rehydrated bloodstain extracts.

The first method we tested was ammonium sulfate(AS) precipitation guided by protein isoelectric points. The plan was to first adjust the solution pH close to hemoglobin’s isoelectric point, pI ≈ 7.1 [4] to reduce its net charge, thereby promoting its selective precipitation upon ammonium sulfate addition. After pelleting hemoglobin, the supernatant would then be acidified toward albumin’s isoelectric point, pI ≈ 4.7 [5], enabling the subsequent precipitation of albumin.

However, this approach failed in practice. Despite attempts to steer the pH in both steps, the system remained buffered around pH 6 throughout the process. As a result, both hemoglobin and albumin were detected in all collected fractions, indicating that neither protein had been selectively removed.

Spectrophotometric measurements taken at 420 nm and Bradford assay indicated that both hemoglobin and albumin were present across all collected fractions, suggesting poor separation. SDS-PAGE revealed a 15 kDa- hemoglobin monomer - band in all fractions.

During the meeting, Wittfooth reviewed the strategy and made several key observations:

• She confirmed that ammonium sulfate fractionation is highly nonspecific and sensitive to multiple factors, including precise saturation levels, and pH.


• She confirmed that the 15 kDa fraction was the hemoglobin monomer.


• She noted that ion exchange chromatography would in principle be better suited to separate HSA and Hb due to their differing pIs, but understood that this was not feasible given the lack of resources.


• Affinity purification using antibodies and size-based separation techniques were briefly discussed but dismissed, mostly due to conceptual conflicts: our detection system itself relied on immunological specificity. From what we had learned from our research, we suspected that aging the blood stain would lead to yields that would be inversely proportional to the age of the blood stain.


• She verified that albumin could be precipitated, as long as the process was reversible and did not disrupt structural integrity.


• She emphasized the importance of tight buffer control, recommending a Tris concentration of at least 50 mM to stabilize pH and avoid drift during salting out or heating.

As an actionable next step, we decided to initiate heat-assisted fractionation experiments in combination with AS and pH-controlled buffers, to improve the separation of hemoglobin without compromising albumin. The session clarified the underlying causes of earlier separation failures and helped us set more realistic expectations for matrix purification within our available means.

Professor Kaisa Linderborg

We contacted Professor Kaisa Linderborg of food chemistry at the University of Turku in spring 2024 she supported the ABOA iGEM team by helping us plan and implement laboratory work involving human blood samples in accordance with the biosafety procedures of the food sciences unit.

In early May,our team contacted her to request a hepatitis B vaccination. Although working with blood samples would have been technically possible without it, only two members of our lab team had been vaccinated at that point. This would have placed an unsustainable burden on the remaining person if the other became unavailable. With a third vaccinated member, our lab work became far more resilient and practical. Linderborg supported the request the same day and provided the necessary referral form along with a document on how to safely handle biological samples.The form was collected in person in mid-May. During the same meeting, Linderborg reviewed the biosafety document point by point with us to ensure that the content and intent of each instruction were fully understood.

From late May to early June, she continued to support the team via email. She responded promptly and clearly to questions about how to interpret the biosafety document in specific scenarios — such as when no dedicated centrifuge was available. Her input helped us ensure that the planned lab work could proceed safely, responsibly, and in compliance with the guidelines.

Biotechnology Department

Before the meeting, we had conducted an initial trial of recombinant fusion protein expression, periplasmic extraction, and IMAC purification based on our first version of the protocol. Initially, we contacted Docent Tuomas Huovinen. We gave him details of our proteins, strain, used protocols, and experimental results. Our primary concern was troubleshooting, as we had successfully purified only 1 of 10 target proteins, and one periplasmic extract out of 10 did not contain any protein, with three exhibiting minimal protein presence based on visual assessment of Bradford assay.

As he was on his summer leave, Huovinen didn’t have time to get properly acquainted with our experiment, but he suggested doing lysis by lysozyme and freeze–thawing method instead of periplasmic extraction as it in his experience could increase the yield. Another idea he had was doing measurements with the lysates to account for typically low expression levels of fusion proteins and anticipated losses during IMAC purification.

Even though we got valuable suggestions for future attempts from Huovinen, we still felt like we needed extra assistance. We contacted PhD Aapo Knuutila, who had assisted us previously with various matters, and he recommended contacting doctoral researcher Sami Oksanen and PhD Eeva-Christine Brockmann while he was also on his summer leave. Thus, we ended up scheduling a meeting with Oksanen and Brockmann.

During the meeting we expressed our issues with the practical side of the expression and purification parts of the protocol. For purification especially, we had a long list of ideas on how to improve the protocol, but were unsure what we should prioritize as our time in the lab was limited. Essentially, we wanted people with more expertise than us to listen to our ideas for improvement, comment on them and give their opinion on what is reasonable to do in the limited time frame we have.

Brockmann and Oksanen agreed with Huovinen that trying lysis is a worthy endeavour. We expressed concern regarding the reagents needed for the lysis by lysozyme and freeze–thawing method as they were not available for us at the moment. Lysis by sonication was then suggested as an alternative as it doesn’t require reagents as long as the solutions are kept cool.

With our first attempt at expressing our proteins, we had come across an unexpected struggle of not having shakers with cooling functions. Thus, we couldn’t grow the expression cultures in 18 °C for 20 hours as per protocol. Instead, the expression culture was grown at about 21 °C for 18 h. Oksanen and Brockmann reassured us that the 18 °C suggested by the protocol is actually quite an extreme precaution, and that incubation temperatures slightly above this threshold are unlikely to cause any significant impairment.

We got a suggestion to grow the expression culture at 26 °C for less than overnight as in BL21(DE3)pLysS overnight cultures grown at 26 °C the protein often starts to leak from the periplasm. 8-9 hours was proposed as optimal but it was soon agreed that 10-12 hours would be the most practical.

Furthermore, we considered lowering the concentrations of IPTG that was used for induction from 1 mM to more typical levels. 0.1 mM, 0.2 mM or 0.5 mM were assured to be sufficient. To increase protein expression efficacy, adding 0.5 % glucose to preculture and 0.05 % glucose to expression culture were proposed. The cells don’t start expressing the proteins before all the glucose has been used up which has led to greater yields.

We expressed concern that 1 mM EDTA in our TSE buffer used for periplasmic extraction by osmotic shock method could chelate essential metal ions, potentially impairing subsequent IMAC purification. We were reassured that the concentration is sufficiently low to mitigate this risk, though it might still influence overall yield. However, using Benchling to calculate the pI values of our proteins was suggested because Oksanen had noticed that some of our proteins had pIs close to 8, the pH value of our TSE buffer, or 7, the pH value of our wash and elution buffers for IMAC. Thus, low yield was to be expected of those proteins as they are not soluble in buffers that have pH near their pI. It was suggested to modify the pH of the buffers so it would be ± 0.5 the pI values.

For IMAC, we had used aged Co-NTA resin without regeneration due to unknown usage history and reagent access constraints. We expressed the idea of using Ni-NTA resin, which we also had access to, because it gives better, though less pure, yields. Continuing to use Co-NTA resin was however encouraged as the sole purified protein, Nb77-SmBit, was highly pure according to SDS-PAGE.

We also had to use the batch spin method because we did not have access to columns. The batch spin method was time consuming and also cost us a lot of pipette tips which we had a limited amount of. We learned that, however, the batch spin method actually has an advantage over the column method: it gives more time for His-tagged proteins to bind.

The possibility of imidazole-induced protein precipitation was raised, which had not previously been considered. Oksanen proposed that we check if our protein solutions contained any “snowflakes” as that would be a sign of precipitation. Luckily, they did not, but it was in any case interesting to learn of a possible problem that we had not thought of previously.

We came to the meeting prepared with our own ideas of potential problems with our attempt. However, getting to properly explain our own ideas and brainstorm new ideas as well proved immensely helpful . Many of the things we had previously perceived as bigger issues with our attempt were actually pretty easy to solve with the help we got from Oksanen, Brockmann, and Huovinen. We got introduced to new potential problems researchers might face as well. Based on the suggestions we received through the discussion we had with Huovinen, Oksanen, and Brockmann, we created version 2 of the protocol for protein expression, periplasmic extraction, and IMAC purification. You can read more about protein expression, periplasmic extraction, and IMAC purification related DBTL cycle here.

Ethics

Professor Matti Häyry

As our team began shaping the concept for our project, we quickly recognized that forensic technologies raise important ethical questions. Our goal from early on was to create a rapid on-site detection test to aid crime scene investigations. However, we wanted to ensure that our test would not cross into the territory of profiling, which would raise concerns about privacy, bias, and misuse.

To explore these issues, we reached out to Professor Matti Häyry, a Professor and a Senior Fellow at Aalto University, whose academic expertise include philosophy, ethics, and synthetic biology ethics. With his knowledge, we aimed to gain an external ethical perspective to ensure that our work would align with responsible scientific practices.

In our email exchange during June (weeks 23 and 24), we discussed the general ethics of synthetic biology to have better science communication and to better understand the potential risks related to our project. Häyry supported our choice of creating a non-profiling tool, highlighting that even without identifying individuals, our test could provide real value by speeding up investigations without the downsides associated with profiling.

We also raised concerns about the possibility of misinterpretation or misuse of the test results. Häyry encouraged us to aim for a foolproof design that minimizes the risk of misuse even when misused intentionally. He also noted that we seemed to be on the right path towards this. This led us to consider the need for clear usage guidelines that define the limitations of our test and emphasize the bordercase situations where it should not be used to draw definitive conclusions.

We combined this idea with the legal principle of “beyond a reasonable doubt”, which we had previously discussed with Kimmo Nuotio. Together, these perspectives helped us shape the ethical framework for our project and informed both the design and communication strategies.

Häyry also raised a question regarding the necessity of our test and whether its potential benefits would justify the costs associated with its development. Fortunately, we had been addressing this concern throughout the project.

In the beginning of our project we consulted forensic chemists, who affirmed the value of our proposed test and encouraged further development. During the design phase, we conducted interviews with police officers and crime scene investigators, who not only confirmed the usefulness of our test but also revealed that a solution like ours has been wished for in the field.

Even if our test does not result in a fully deployable product by the end of iGEM, our conversation with Mari Humalajoki emphasized an equally important outcome: the broader impact on the area of forensic research. Our project brings attention to the potential of open, university-driven research in a field that, in Finland, is traditionally confined to the closed environments of the National Bureau of Investigation. By working in an open academic setting, we contribute to increasing transparency and collaboration in forensic science.

Doctor Marko Ahteensuu

On the 2nd of July, we had a meeting with Doctor Marko Ahteensuu, a docent at the University of Turku with more than twenty years of experience in bioethics and medical ethics. His background and ongoing engagement with ethical issues in science, particularly in areas such as biosafety, genetic technologies, and public participation, made him an excellent expert to consult for our project.

We reached out to him to ensure that our project not only met regulatory standards but also addressed broader ethical considerations from the outset. Through the discussion, we learned that while compliance with legislation is the minimum ethical requirement, projects like ours also carry a broader responsibility to anticipate potential societal concerns and unintended consequences. Ahteensuu emphasized the importance of transparency, clear communication, and building trust through open dialogue. He also mentioned how public perceptions, especially regarding the use of genetic or biological information, can shape the acceptance of new technologies. This was particularly relevant to our project, as it involves analyzing biological samples in a law enforcement context, which can raise concerns about privacy, data use, and possible misuse.

During our conversation, we also asked Marko about synthetic biology ethics and the responsibilities of researchers in developing a new tool. Ahteensuu stressed that in the context of synthetic biology, ethical questions often center on biosafety, unintended consequences, and the boundary between what is considered living and non-living. Although commenting on moral responsibility, he added that the responsibility of researchers should be understood as covering the entire research process, from early planning through to publication and application. This includes considering potential misuse, such as dual-use scenarios where research could be repurposed for harmful ends, and taking reasonable steps to prevent such outcomes. Ahteensuu noted that it is already a significant contribution if researchers thoughtfully identify risks and integrate safeguards wherever possible. He highlighted that the work we do to ensure that our test is alongside regulatory standards and mitigates the risk of misuse, even if such misuse is unlikely, is valuable and essential.

The insights we gained from this meeting prompted us to ensure that our test is used strictly within legal and ethical boundaries, and to consider how we might clearly communicate the limitations and intended uses of our technology to stakeholders and the public. Ahteensuu’s perspectives helped us recognize the importance of engaging with ethical questions proactively, not only to avoid harm but also to strengthen the societal value and credibility of our forensic innovation.

Other

Manuel Blank (iGEM Dundee 2015)

To discuss taking part in iGEM with a forensic science project, we contacted Manuel Blank from the University of Dundee 2015 team to have a Zoom interview on the 3rd of April. We had a long list of questions and a short presentation of our work so far prepared.

After presenting our work, we started by discussing more practical questions related to working with the forensic science subject. Since the University of Dundee team with Blank participated in iGEM a decade ago, his recollection of some of the details was understandably fuzzy. One key point Blank emphasized, however, was to design the product so that it can not be misused. Forensics is a field of science where the effects of misuse can be especially harmful.

We moved on to talking about what kind of reception their project received and why forensic science is so rarely seen as a subject in iGEM. Blank recalled receiving a lot of positive feedback from both judges and other contestants since the project’s subject was so unusual in iGEM. Blank mentioned that the forensic science theme made branding and marketing easy for the project.

The team took a light-hearted approach with their wiki, leaning into the crime show theme with, for example, their project name, CSI: Dundee, and their team page where the team members were dressed as famous pop-culture detectives or villains. We asked him how their light-hearted theme was received, as the subject of the project is quite serious. The reactions were funny but positive, according to Blank. He could not recall exact feedback from the judges, but it was overall very good, which is also reflected in the gold medal the team received for their work.

Regarding judging, Blank mentioned that to help the judges assess the project, they made a subpage on their wiki where they explained briefly how they fulfill the criteria for the gold medal. This was an ingenious idea that our team plans on implementing in our wiki.

Next, we moved on to talking about the human practices and outreach work the team did. The University of Dundee has a large forensic science department, which helped the team in finding insightful stakeholders. Blank emphasized that casting a wide net with human practices work is important to get as many useful perspectives as possible. Contacting less obvious stakeholders, such as crime authors and media outlets could be interesting.

The CSI: Dundee team did not pursue the education special prize, but we wanted to know if Blank had any ideas on how it could be incorporated into the forensic science theme. Blank recommended focusing on blood itself as an aspect of our project that is more appropriate for younger audiences. He recommended we think about a slightly older audience if we want to maintain forensic science as the core of our education work. Blank suggested the age bracket when kids start watching crime shows such as CSI as a starting point.

The discussion moved to what makes a successful iGEM project next. Blank emphasized that people and project management are a huge aspect of these kinds of group projects. Good science is, of course, necessary, but good people are more important. The CSI: Dundee team had a weekly meeting every Monday morning to make sure everyone was up to date and to split tasks since the team had split into smaller subteams. Blank also mentioned that the team did other fun stuff together unrelated to iGEM to build interpersonal connections. The team needs to work well, which is a lot easier when people get along.

Lastly, we asked for anything he wished he knew at the beginning of the project and other tips. Starting the wiki early was the first thing Blank brought up. Working on the wiki takes a lot of time, so the earlier the better.

Additionally, Blank re-emphasized that iGEM is a lot more than a science competition, with the non-science parts being shockingly labour intensive. Also, making sure everyone is on the same page and getting regular feedback for their work is critical.