Abstract

Forensic science is a field that lacks reliable methods for determining the age of bloodstains, a key factor in reconstructing timelines for crimes. Currently, the investigation relies heavily on visual assessment of bloodstains, which is dependent on personal interpretation. To address this issue, the ABOA 2025 team is developing a new, innovative, on-site forensic investigation tool – VeriFied – that allows crime scene investigators to quickly and reliably assess the age of bloodstains. The method is based on an enzyme fragment complementation assay that produces a bioluminescence signal. The signal intensity correlates with the levels of oxidized human serum albumin that increase over time as bloodstains are exposed to air. Our aim is to introduce a scientifically robust, quantitative technique that reduces reliance on individual interpretation and improves the accuracy of investigation done at a crime scene. This enables criminal investigators to establish more precise timelines and identify relevant bloodstains to the case, guiding the investigation with a scientific basis and solving crimes that would have previously gone unsolved.

Background

Inspiration and motivation

In modern forensic science, consistent, objective, and quantitative methods are often considered the gold standard for criminal evidence [1], [2]. Techniques such as DNA short tandem repeat (STR) analysis, immunoassays for toxicology, and chemical mass spectrometry analysis are commonly used in criminal investigations and widely considered reliable in court [3]. Even though no evidence can be regarded as absolutely objective, some can be considered essentially objective due to their high accuracy and minimal influence from personal feelings or opinions. On the other hand, the interpretation of results is always subjective. Currently, a large amount of evidence used in court is based on methods such as fingerprint analysis and eyewitness testimony that have been proved highly subjective and untrustworthy [4], [5].

Forensic science is an underrepresented field in iGEM and synthetic biology in general. A big inspiration for our team was the University of Dundee 2015 team with their Forensic Toolkit. Their kit was designed to estimate the age of latent fingerprints, differentiate a variety of biological stains from each other, and detect trace amounts of chromate in bone [6]. Overall, the field of forensic science seems to be largely overlooked by synthetic biology researchers, even though there is huge potential to help expedite investigations, allow the use of new types of evidence, introduce improved ways to analyze evidence, and save resources with new synthetic biology innovations. Based on our interviews, at least in Finland, most of the research done in the field is conducted outside of universities in other governmental agencies, such as the Finnish National Bureau of Investigation. This limits the amount of information available to the general public.

Bloodstain age analysis

At the crime scene, calculating the time since deposition (TsD) of a biological stain can help piece together a timeline of the events, steer the investigation, and help determine the bloodstains relevant to the crime Based on interviews we have held with Finnish crime scene investigators and crime chemists, the timeline of a crime is currently assembled from miscellaneous evidence such as the internal temperature of a corpse, when mail was last opened, surveillance footage, and phone use.

Evidence related to the time of the crime is also critical during the court proceedings. Determining the time of the crime can prove or disprove alibis and help figure out who could and could not have been at the crime scene during the events of the crime. Additionally, information that can be used to determine the time of the crime can help to confirm or deny the validity of other evidence. For example, if a witness testimony does not match the timeline determined by other evidence, other statements from the witness may lose credibility.

In crimes such as missing persons cases, speed is often a crucial aspect of the investigation. For this reason, and to save resources, on-site testing is a rising trend in forensic science [11]. Especially on-site tests that crime scene investigators are able to perform quickly, with minimal training, and without bulky equipment are in high demand.

The only methods used for estimating the TsD of a bloodstain rely on visual cues such as dryness and changes in coloration visible to the naked eye. A large variety of techniques have been devised, but they all face issues related to practicality, price, and most often accuracy and precision [7], [8]. Many techniques are simply not accurate enough in a variety of crime scenes, so they are generally not applicable for crime scene investigation purposes.

Techniques that have been previously researched include, but are not limited to, enzyme activity measurements, various spectroscopic methods to determine hemoglobin denaturation, RNA degradation, and methods that estimate time of day based on hormones and the circadian rhythm [7]. Many of these methods are confined to a laboratory setting and can not be applied as an on-site test. There is currently no widely used method to objectively analyze the age of bloodstains [7].

How biological stains such as bloodstains connect to the determination of the time of the offence.

In addition to being a crucial piece of evidence for reconstructing a timeline of the events, the TsD of a bloodstain can also be used to filter out irrelevant stains from further analysis, such as DNA sequencing. Multiple countries are facing large backlogs of DNA evidence that are waiting to be analyzed, resulting in delays within the criminal justice system, additional costs, prolonged imprisonment of the innocent, and the guilty avoiding conviction [9], [10]. By determining the relevant bloodstains early in the investigation, criminal investigators can avoid sending irrelevant samples for further expensive and time consuming analysis in a forensic laboratory. Currently, the forensic laboratory is forced to analyze all samples as if they were relevant to the crime, even if many of them are not.

How the VeriFied test can be utilized in pre-trial investigations and judicial proceedings.

An obvious but relevant comparison can be drawn between on-site testing in forensic science and point-of-care testing (POCT) in healthcare. POCT can provide benefits such as faster treatments, cheaper tests, and reduced labour costs [12]. Although beneficial in many ways, care must be taken in designing POCT or on-site tests and deciding when and how to deploy them due to issues with environmental conditions and errors that may occur when they are used by untrained individuals [13]. This is why on-site tests should be designed to be as simple as possible, with minimized decision making, either being largely automated or containing very simple instructions [13]. Additionally, tests should be validated to take into account margins of error and environmental factors [13].

After discussions with various experts on forensic techniques, one of the most important aspects to keep in mind while designing an on-site test is to make sure the test will not cause contamination at the crime scene. This means that ideally the test is cell-free and free of nucleic acids and as many other contaminants as possible that could cause issues with other testing done at the crime scene. In addition, the test should be nondestructive to allow further downstream tests, such as STR and toxicology analysis on the same sample if possible. Due to the values of our team, we additionally decided that a nonprofiling test would be the best solution ethically. Talking with stakeholders later in the project reaffirmed this choice.

Comparison of on-site tests and tests confined to the laboratory.

Requirements of the VeriFied test based on literature research and discussions with stakeholders.

Solution

The ABOA 2025 team aims to develop a novel solution for bloodstain age analysis with synthetic biology. VeriFied is an on-site test that allows crime scene investigators to determine the age of a dried bloodstain based on bioluminescence signal intensity directly at the crime scene. In dried bloodstains, albumin oxidizes due to the presence of oxygen. The bioluminescence signal intensity decreases in correlation with albumin oxidation. Measured with a handheld device, older bloodstains will give a weaker signal compared to fresher stains.

The workflow of the VeriFied test.

Human serum albumin and oxidation

Bloodstains contain a helicoidal protein called human serum albumin (HSA), which is the most abundant protein in plasma, representing half of the total protein content. HSA concentrations vary between 3.5 g/dL to 5 g/dL in healthy individuals [14]. Only containing 585 amino acids and being 66 kDa, HSA is a relatively small globular protein [15]. HSA modulates the oncotic pressure of plasma, and it also transports both endogenous and exogenous ligands such as drugs, fatty acids, and bilirubin [14]. HSA also has antioxidant and anti-inflammatory properties [16].

As seen in Figure 1, HSA consists of three domains (I, II, and III), each of which contains two subdomains (A and B) [16]. HSA has 35 cysteine residues in total, all but one of which participate in intramolecular disulphide bonds. Cys34 remains the only free cysteine residue in HSA, which makes it prone to attack of reactive species [17]. Cys34 accounts for 80 % thiols in plasma and is capable of undergoing both thiolation and nitrosylation [15].

Figure 1. Human serum albumin (PDB: 1AO6). Blue: domain I, yellow: domain II, red: domain III, cyan: Cys34. Figure made in pyMOL.

HSA appears in three main oxidation states in human blood, determined by the redox state of Cys34. When Cys34 is in its reduced form, HSA is called mercaptalbumin or HSAred. In this form, Cys34 has a free thiol group. In healthy adults, about 70-80 % of HSA exists in its reduced form [16].

Oxidized forms of HSA include nonmercaptalbumin1 (HNA1) and nonmercaptalbumin2 (HNA2), collectively known as HNA or HSAox. In HNA1, Cys34 forms a disulphide with compounds like other cysteines. Thus, HNA1 can also be called mixed disulphides. 25 % of Cys34 appear as mixed disulfides. A small fraction of Cys34 is highly oxidised to sulphinic or sulphonic acid. This group is called HNA2. Cys34 can also be nitrosylated by nitric oxide, though only nanomolar amounts have been reported in vivo [16].

Different oxidation states of human serum albumin. Alb = human serum albumin, HCys = homocysteine, G = glutathione. Figure adapted from [17].

Under normal physiological conditions, the concentration of HSAox relative to HSAred is low. However, during disease processes characterized by oxidative stress, HSAox formation increases. HSAox concentration increase is linked to diseases such as diabetes mellitus, liver diseases, and coronary artery disease. Aging, intensive exercise, and invasive surgery can also increase HSAox concentrations [16].

Oxidation has been proven to cause structural differences in HSA. HNA1 becomes more susceptible to tryptic proteolysis because S-cysteinylation causes conformational changes in HSA that make the cleavage sites more accessible. Additionally, oxidation affects the structures of the domains differently. S-cysteinylation changes the conformation of the entire domain I as well as the domain I/II interface. The redox state of Cys34 has also been shown to affect binding affinities [18].

On-site test for bloodstain age analysis

Our on-site test uses HSA as a biomarker for bloodstain age determination. The abundance of HSA in blood and the variety of HSA binding nanobodies available make it a great biomarker for our purposes. Our hypothesis is that HSA oxidises as dried bloodstains are exposed to oxygen in the air. We are able to detect HSA from bloodstains with nanobodies that bind to it. Nanobodies are structurally similar to human IgG heavy chains but are derived from camelids’ heavy-chain-only antibodies [19]. Nanobodies are characterised as small, ranging from 12-14 kDa, water soluble and stable antigen binding fragments. They also have a high antibody binding affinity [20].

Because of nanobodies’ versatility, small size, and specificity, we were able to choose high affinity nanobodies that specifically bind to oxidation prone and oxidation stable HSA epitopes. In a study by Shen et al., they were able to identify thousands of high affinity anti-HSA nanobodies for drug delivery and map HSA nanobody epitopes of these nanobodies [19]. All but one nanobody used in our project have been studied by Shen et al.

Our test uses two different nanobodies at a time (Figure 2). These nanobodies bind to different epitopes on HSA: one binds to an oxidation stable epitope while the other binds to an epitope whose 3D structure changes upon HSA oxidation. The control test, on the other hand, also has two nanobodies, but in contrast to the actual test, these nanobodies both bind to oxidation stable epitopes. The control test indicates overall HSA concentration, while the actual test tells the concentration of reduced HSA. We used data provided by Kawakami et al. to define areas of HSA that are prone to oxidation induced conformational changes [18]. We combined that information with the nanobody epitope data provided by Shen et al. to choose the best nanobodies for our test design [19]. Both of our nanobodies are connected to a split enzyme system via a flexible amino acid linker.

Figure 2. Human serum albumin (PDB: 1AO6) with bound Nb29 and ALB8. Blue: domain I, yellow: domain II, red: domain III, cyan: Cys34, green: ALB8 (PDB: 8Z8V), orange: Nb29. Figure made in pyMol. Amino acid sequence for Nb29 is from the article by Shen et al. [19]. Homology modeling of Nb29 was done in SwissModel. Docking of Nb29 to HSA was done in HADDOCK.

Our team chose to use NanoLuc based NanoBiT as our split reporter enzyme. NanoLuc’s advantages are its small size and bright luminescence. NanoBiT contains two subunits called SmBit and LgBit, SmBit being significantly smaller (1,3 kDa) than LgBit (18 kDa). NanoLuc reacts with its substrate furimazine and produces bioluminescence, which can be measured on-site with a portable reader [21].

However, because NanoLuc has been divided into two parts, the fragments exhibit only a little reporter activity separately. NanoLuc fragments regain activity when they are brought together, but that does not happen naturally as the two parts have a low affinity for each other. Thus, NanoLuc stays inactive until both of our nanobodies are able to bind to HSA, and bring NanoLuc parts into a close enough proximity with each other with the help of the (GGGGS)n linker [21].

An animation depicting the enzyme fragment complementation assay behind the VeriFied test.

Nanobodies are able to specifically bind to HSA in bloodstains, but their binding properties are directly caused by HSA’s specific structure. During oxidation, the 3D structure of HSA changes, which results in the nanobodies’ weakened affinity for HSA. This will result in a weaker bioluminescence signal, because now the NanoLuc parts are not able to form a functional enzyme. Thus, with our test, crime scene investigators are able to analyse bloodstain age based on the bioluminescence signal intensity. The stronger the signal, the fresher the stain is.

As always, new methods require calibration. Our lab team has taken up the task to test our proof of concept. We are working with bloodstains of different ages to check the signal intensity differences to accurately report our test’s ability to differentiate bloodstains of different ages. Without these experiments and the results these will yield, it is hard for us to make any promises about the abilities of our test. However, based on limited research data available, it seems like 2 weeks is a significant time for HSA degradation in bloodstains. In a study by K. Rajamannar, HSA was not detectable in bloodstains after 2 weeks by immunoelectrophoresis [22]. As our test is based on a different method, it is hard to say if the 2 weeks timeline translates to our project as well. Thus, we are using multiple methods to measure HSA oxidation.

Levels of potential accuracy for the VeriFied test.

Our mission

Our mission is to revolutionize on-site forensic investigation with VeriFied, a novel diagnostic tool for estimating bloodstain age directly at the crime scene. This fast and easy-to-use test will allow forensic investigators to identify relevant bloodstains with minimal equipment and without costly and time consuming laboratory analyses. Our test aims to provide objective, unbiased, and nonprofiling results, which would help in determining the timeline of a crime and identifying the bloodstains relevant to the case. Importantly, it is a nondestructive method, preserving the sample for further forensic analyses.

VeriFied is the first of its kind and uses HSA oxidation to determine the time that has passed since the deposition of a bloodstain. Our initial goal is to establish a proof of concept for our invention under stable laboratory conditions. Environmental factors such as humidity, temperature, and even bloodstain size are likely to affect the rate of HSA oxidation. Therefore, further calibration and validation must be done to identify the conditions so that VeriFied can provide reliable and reproducible results.

In addition to developing our test design, we aim to contribute to HSA oxidation research. This is not only relevant for forensics, but holds significance in medical research, including the study of diseases caused by oxidative stress and the transport chain for HSA for infusions [23].

In the future, we hope that VeriFied will develop into a crime scene investigation tool that can make a difference in the way blood samples are analysed and prioritized. Eventually, we envision VeriFied becoming a widely adopted, field ready tool that can help in solving crime cases more accurately and effectively. After the first stages of development, VeriFied could be enhanced by incorporating additional time sensitive biomarkers. This could improve the test’s precision and accuracy in a wider variety of environmental conditions.

We believe that synthetic biology innovations can make a great difference in the acquisition and analysis of biological samples at the crime scene. Through our project, we hope to promote research and innovation in this field, both in iGEM and the overall scientific community.