Abstract

Tattoos have long served as markers of identity and belonging. InkSight repurposes this historical medium for modern medicine as a platform comprising engineered mammalian cells encapsulated in a hydrogel matrix for intradermal delivery as a tattoo. This system enables the real-time, equipment-free detection of circulating biomarkers through direct interrogation of the interstitial fluid. The engineered cells express a Modular Extracellular Sensor Architecture (MESA) for target recognition, which triggers one of three engineered contrast-change mechanisms: expression, assembly, or agglomeration. This response is driven by synthetic melanin-producing compartments, specifically encapsulin nanocages that house tyrosinase to enable localized and safe melanin production, resulting in a visible shift in tattoo contrast. InkSight constitutes, to our knowledge, the first paradigm for a continuous, cell-based tattoo biosensor with a visible output, establishing a foundational platform for decentralized diagnostics and personalized medicine.

Fig. 1. / Overview of InkSight project. A living biomedical tattoo that can signal physiological change is injected into the epidermis. The tattoo ink contains engineered mammalian cells that are embedded in a hydrogel matrix for safety reasons. If a specific biomarker is present in the interstitial fluid, the cells change their contrast, which is mediated by synthethic melanosomes producing melanin pigment.

Introduction

The human skin has long been a canvas for identity, storytelling, and belief. Across cultures and centuries, tattoos have inscribed a visual language onto the body, preserving and communicating everything from beauty and social status to medicinal practices and spiritual protection (Krutak, 2015). This enduring history is exemplified by a rich diversity of traditional techniques, each deeply embedded in its cultural context.

Want to learn more about the usage of tattoos in ancient medicine?

The use of tattoos in medicine is not a modern innovation, but ancient practice with a deep history. The world’s oldest evidence of medicinal tattooing comes from Ötzi the Iceman, a glacier mummy discovered in the Alps 3250 B.C. His well-preserved skin reveals a series of line-based tattoos on his lower back, ankles, and wrists. According to tattoo anthropologist Lars Krutak, 80% of these tattoos correspond to traditional Chinese acupuncture points, suggesting they were a prehistoric therapy for ailments like rheumatism and gastrointestinal issues (Scallan, 2015). This tradition, also documented in the illustration of tattoo designs seen on a Sinaugolo person from British New Guinea, adapted from The Journal of the Anthropological Institute of Great Britain and Ireland, and licensed unter CC-BY-4.0 (Creativecommons.org, 2025), demonstrates that the concept of the skin as a site for therapeutic intervention is thousands of years old.

Across Southeast Asia, the ancient hand-tapping method creates the sacred sak yant tattoos, where spiritual motifs are ritually inscribed into the skin for protection and luck. In Japan, the meticulous tebori technique employs handheld needles to craft intricate, symbolic designs like dragons and koi fish, a testament to patience and artistry. The Polynesian tatau, applied with a tapping tool, tells stories of heritage and social status through powerful geometric patterns. Similarly, the Berber tradition of henna uses a natural paste to create temporary, celebratory body art for weddings and festivals. More than mere decoration, these methods are vital cultural vessels, preserving ancestral knowledge and artistic expression (Medhi, 2023). Their continued practice today highlights a profound, universal human desire to use the skin as a canvas for meaning — a principle that directly inspires the next frontier of tattooing in medical science (Gamaleldin and Orzan, 2023).

(Picture adapted from Collection, n.d.)

Nowadays, tattooing has become a widely accepted form of self-expression and body art in many parts of the world. This change reflects shifting social norms, technological improvements that enhance safety and precision, and the growing visibility of tattoo culture in media and popular art. Nevertheless, in some settings, tattoos may still be viewed with caution or disapproval, particularly in workplaces or institutions where visible body art remains uncommon. Although perceptions of tattoos still vary across cultural and professional contexts, they increasingly occupy a space that bridges art, identity, and function.

Building on this deep-rooted human practice, the concept of tattooing has also evolved within the medical field, where modern applications serve restorative and therapeutic purposes, such as reconstructive micropigmentation and other healthcare-related uses.

Want to learn more about the usage of tattoos in modern medicine?

A prominent application of medical tattooing is scalp micropigmentation, which addresses advanced male pattern baldness and alopecia. This technique involves depositing pigment into the scalp to simulate the appearance of natural hair follicles, creating the illusion of a closely shaved head and camouflaging scars. (Becker and Cassisi, 2021)

Another form of micropigmentation offers a restorative solution for vitiligo, a condition characterized by the loss of skin pigment. The primary goal is to reintroduce pigment into depigmented patches, carefully matching the surrounding skin tone to create a more uniform appearance. The central challenge lies in achieving a precise and lasting color match that blends seamlessly with the patient’s natural complexion, helping to restore the skin’s aesthetic continuity. (Becker and Cassisi, 2021)

Another effective treatment for hypopigmented scars that are not suitable for surgical or laser revision. The restorative goal is to reintroduce pigment into the pale scar tissue, allowing it to blend seamlessly with the surrounding skin tone. This procedure has been shown to yield high rates of patient and physician satisfaction. Recent studies, which involve systematic outcome assessments and collaborative professional input, suggest that medical tattooing can significantly improve cosmetic outcomes after a scar has reached its maximum recovery from other medical interventions. For scars that are hyperpigmented, texturally uneven, or large, a restorative approach is often insufficient. In these cases, the objective shifts to concealment, where an aesthetically designed image or pattern is tattooed over the scar to effectively camouflage it. (Becker and Cassisi, 2021)

Beyond these applications, medical tattooing also plays a crucial restorative role in specialized reconstructive procedures. A prominent example is the recreation of the nipple-areola complex (NAC) following mastectomy and breast reconstruction, where pigmentation is meticulously applied to achieve a natural, three-dimensional appearance. Similarly, micropigmentation is used for toenail restoration, where pigment is applied to the nail bed to simulate the look of a natural toenail for patients who have lost nails due to trauma or medical conditions. These diverse applications underscore the versatility of medical tattooing as a powerful tool for restoring both form and confidence in the wake of medical treatments.

While these medical tattoos, in these cases also called permanent makeup (PMU), provide static information or restoration, a new frontier is emerging with the advent of biosensing technologies. For instance, recent innovations like single-use microneedle (MN) patches have demonstrated a painless method for depositing tattoos that can encode medical data, such as QR codes or ultraviolet-visible symbols, for patient identification and monitoring. (Li et al., 2022)

Current wearable biosensors, such as smartwatches, glucose monitors, and pulse oximeters, have transformed preventive medicine by enabling continuous, mostly non-invasive health monitoring. These devices provide real-time data on physiological parameters, allowing users and clinicians to track recovery, detect anomalies, and support early diagnosis from home. The main functions for wearables can be categorised into monitoring, screening, detection and prediction (Canali et al., 2022). They offer significant benefits such as cost-effectiveness, compact size, and high sensitivity, as demonstrated by widespread glucose monitors for diabetes.

However, their sensing capabilities remain largely limited to physical and electrophysiological signals like heart rate, ECG, or movement (Guk et al., 2019). Despite their benefits in comfort, portability, and cost-effectiveness, most still depend on rigid materials, clinical infrastructure, and frequent in-person appointments, which constrain long-term, seamless monitoring (Mukherjee et al., 2025).

Our technology builds upon this powerful concept of continuous, user-friendly monitoring but introduces a critical, missing dimension: direct molecular insight. By moving the biosensor directly into the dermis, we go beyond tracking heart rhythms to detect specific, dynamic biomarkers inside the body. This offers a bio-chemical window into physiological states, a new class of diagnostics that remains out of reach for current consumer wearables, yet retains their key advantages of at-home usability and comfort after a single, painless application.

This vision of the skin as a diagnostic interface is being actively realized through synthetic biology. The foundational work by Allen et al. (2024) established the core engineering framework, demonstrating that bacteria encapsulated in hydrogel beads could be tattooed into the skin to function as living analytical tools, sensing a broad range of biochemical and biophysical cues. Building on this, the study by Tastanova et al. (2018) provided a critical in vivo proof-of-concept. They moved beyond a sensing platform to a functional diagnostic, engineering mammalian cells to detect a specific cancer-associated condition (hypercalcemia) and report it through the production of melanin, creating a visible, quantifiable biomedical tattoo in a live animal model.

Expanding on these advances, our project, InkSight, aims to develop dynamic, living tattoos that function as in-situ diagnostic monitors. Instead of relying on inert or injected pigments, InkSight utilizes engineered mammalian cells designed to detect specific biomarkers in the interstitial fluid. Their intracellular signaling cascade triggers a visible contrast change in response to altered biomarker levels, in the form of increased melanin production. This establishes a biological “silent alarm” within the skin, visually alerting the user when their physiological balance is off. By integrating synthetic biology with the ancient canvas of the skin, InkSight transforms the tattoo from a passive record into an active diagnostic tool.

In the next sections, we describe the main components of InkSight in greater detail, to position our work within existing academic literature and novel diagnostic approaches.

MESA

The selection of the ideal receptor for the detection of a specific target constitutes a fundamental step in any synthetic-biology-based project. Our objective, however, was to develop a general diagnostics platform capable of detecting a wide range of different targets, while requiring only minimal adaptations to the overall system. To achieve the greatest possible degree of modularity, our research focused on cell-surface-bound receptors, consisting of an extracellular Ligand-Binding Domain (LBD), a Transmembrane Domain (TMD) that provides anchorage to the cell membrane, and an Intracellular Effector Domain (IED) capable of activating a downstream signaling cascade. However, the majority of synthetic receptors belonging to this category are accompanied by substantial drawbacks when employed in our conceptualized system. For instance, although Synthetic Intermembrane Proteolysis Receptors (SNIPR) were recently optimized to be activated by soluble ligands, the precise mechanism of action remains to be elucidated. Thus, the adaptation of the ligand binding domain becomes a multifaceted and complex process (Teng et al., 2024). Conversely, Generalized Extracellular Molecule Sensors (GEMS) can be equipped with antibody fragments, thereby conferring a high degree of versatility to the system. However, this configuration also results in the activation of endogenous pathways and transcription networks, an undesired effect that we sought to circumvent in our setup. (Scheller et al., 2018). Finally, after exhaustive research we were able to determine that the Modular Extracellular Sensor Architecture (MESA) represents the most promising receptor choice, due to its high degree of modularity in all relevant domains, including the LBD and IED, its inactivity with respect to endogenous pathways as well as its ability to detect soluble ligands. (Piraner et al., 2025).

Fig. 2. / Overview of different receptors. Illustrated overview of the three receptors Synthetic Intermembrane Proteolysis Receptors (SNIPR), Generalized Extracellular Molecule Sensors (GEMS) and Modular Extracellular Sensor Architecture (MESA) which we considered for our project.

MESA receptors are comprised of two distinct transmembrane proteins, each of which contains the three aforementioned functional domains (LBD, TMD, and IED). In general, their activation is initiated by ligand binding to the LBD of both MESA proteins, which subsequently leads to the dimerization of the receptor. In the first version of the MESA receptor, one of the two chains contained an intracellular protease. Upon receptor dimerization, the protease comes into proximity with its cleavage sequence on the second protein, thus inducing its cleavage and resulting in the release of a cargo into the intracellular space. Said cargo, typically a transcription factor, once separated from the second chain is imported into the nucleus, where it regulates the expression of a specific reporter (Daringer et al., 2014).

What exactly are proteases?

Proteases are a fundamental class of enzymes that catalyze the hydrolysis of peptide bonds, the connections that link amino acids together in proteins. They are primarily categorized by their site of action: endopeptidases cleave internal bonds within a protein chain, while exopeptidases cleave bonds at the ends of chains. Exopeptidases are further specialized into aminopeptidases, which remove amino acids from the beginning (N-terminus) of the chain, and carboxypeptidases, which remove them from the end (C-terminus) (Hooper, 2002).

The most substantial disadvantages of this system are the inefficient incorporation into the cellular membrane and the high degree of ligand-independent activation due to transient dimerization. Both issues can be addressed by optimizing the TMD, a process that can lead to a significant reduction in misactivation and an enhancement in trafficking to the cell surface. In a similar manner, the employment of a split protease, a protease consisting of two distinct inactive protease fragments, each attached to only one of the receptor chains, that regains activity upon reconstitution, has been utilized in the context of MESA to diminish the amount of ligand-independent activation.

Fig. 3. / Overview of different MESA constellations. Illustrated overview of some MESA receptor, TMD and protease combinations, also mentioned in our MESA Designer Software.

Joshua N. Leonard and his team, the developers of MESA, also conducted a series of experiments characterizing a high number of suitable TMDs and the split protease approach for their receptor (Daringer et al., 2014; Edelstein et al., 2020). During the development and testing of our receptors, we sought their counsel on multiple occasions. A thorough description of His indispensable guidance and advice can be found on our Human Practices page. (Learn more about our HP expert meetings!)

Antibody fragments and binding domains of native receptors can both be engineered to construct the MESA LBD. Nevertheless, in our experience, the identification of a suitable LBD can pose a significant challenge, particularly in the context of small molecules without known native receptors. Additionally, in the case of a monomeric target, the LBDs must recognize two distinct epitopes to enable dimerization. The selected LBD may then be combined with countless possible linkers, TMDs, protease variants, and intracellular cargo molecules, each of which fulfills a highly specific function. Confronted with the obstacle of developing and testing multiple receptor designs in a relatively short timeframe, we decided to build MESA Designer --- an intuitive, comprehensive toolkit that transforms MESA receptor design from a weeks-long manual process into a streamlined, error-free workflow that requires only moments before completion. Check out our software page for more!

Upon the binding of a ligand to the receptor, complex signaling cascades are initiated, which ultimately result in a visible change in the engineered cells contrast. The functionality of the system is contingent upon the precise coordination of multiple components, which will be introduced in the following sections.

Synthetic Melanosomes

Melanins represent a prevalent class of bio-pigments, found in all domains of life. Their function is characterized by remarkable diversity, encompassing diverse roles such as camouflage in animals and the stabilization of cell walls in plants. Furthermore, melanins function as a protective agent against various environmental stresses, including UV-radiation, radioactivity, and water scarcity. The various types of melanin are distinguished primarily by the composition of their monomers and the hue of the resulting pigment. We primarily focused our research on eumelanin, a brown-black pigment found in animals, microorganisms and fungi, with a relatively simple biosynthesis pathway, which begins with the amino acid tyrosine and results in a polymer composed of 5, 6-dihydroxyindol (DHI) and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) (Guo et al., 2023). Its simple synthesis, relatively low toxicity and good visibility made it the ideal pigment for our project. Read more about alternative candidates here!

Tyrosinases are copper-dependent enzymes that catalyze two critical reactions in the melanin synthesis pathway, namely the hydroxylation of L-tyrosine to L-DOPA and the subsequent oxidation of L-DOPA to L-DOPAquinone. The ensuing reactions are autocatalyzed and comprise the synthesis of dopachrome, which decarboxylates to DHI or tautomerizes to DHICA. However, depending on the specific biological environment and the presence of additional enzymes and cofactors, the precise monomer ratio, oxidation level and the presence of additional bound compounds may vary (Guo et al., 2023; Kipouros et al., 2022; Kishida et al., 2015).

Fig. 4 / Melanogenesis chemical reaction. Chemical reaction of melanin production, also called melanogenesis, where tyrosinases are involved.

Unfortunately, melanin, or more precisely, melanogenesis, has been shown to possess both cytotoxic and mutagenetic properties, primarily due to the intermediates of the synthesis pathway. Mammals evolved a solution to this problem by generating specialized melanin-producing cells (melanocytes) with dedicated melanin synthesis and storage organelles called melanosomes. These spherical vacuoles are a distinctive type of lysosome-related organelle that undergo a sophisticated maturation process involving both lipids and proteins, culminating in the transfer of the mature stage IV melanosomes to surrounding keratinocytes (Cichorek et al., 2013; Graham et al., 1978; Guo et al., 2023; Raposo and Marks, 2007).

Fig. 5 / Melanogenesis in the cell. Illustrated summary of the four stages of melanosome production.

The exact replication of this intricate process would require an immense engineering effort. Instead, we chose to mimic this strategy by employing bacterial encapsulins so as to create orthogonal compartments to confine melanin production. Encapsulins are bacterial shell proteins that have the capacity to self-assemble into pH resistant and temperature stable nanocages. The triangulation number T indicates the number of identical subunits in a nanocage, typically 60 (T = 1), 180 (T = 3) or 240 (T = 4). Thus, resulting in nanocages with sizes of approximately 18 nm, 32 nm and 42 nm respectively. In their assembled state, the nanocages possess pores measuring approximately 2—11 Å, a size sufficient to permit the diffusion of tyrosine while impeding the release of melanin once it has been synthesized. This raises the question of how to incorporate an active tyrosinase, an enzyme with a size much greater than 2—11 Å, into such a nanocage. Fortunately, a comprehensive set of encapsulation signal peptides has been identified. These tags, if attached to larger proteins, function as shuttle vehicles during the encapsulation process, facilitating the internalization of larger cargo into the nanocages (Sigmund et al., 2018).

T=1 Encap

T=3 Encap

T=4 Encap

Although encapsulation signals address the fundamental problem of targeting peptides and proteins to the assembled nanocages, they lack the capacity to precisely control stoichiometry and prevent melanin synthesis outside of the assembled compartments. In order to address these issues, a series of approaches were developed and subsequently tested, among others with HPLC and Native PAGE, as suggested by Prof. Stefanie Frank from University College London (UCL) during one of our very insightful expert meetings. Firstly, we hypothesized that our engineered enzymes Learn more about how we engineered our enzymes, to better control their activity could be directly fused to the encapsulins, obviating the necessity for target peptides. With this approach we expected enhanced control of stoichiometry and decreased pigment synthesis outside of the synthetic compartments. Secondly, we conceptualized the fusion of sterically demanding domains directly to the N-terminus of the encapsulins, with the objective of impeding the assembly of the nanocage. By integrating TEV cleavage sequences in the region of the linker between encapsulin and the steric hindering domain, we sought to exert control over the assembly process itself. Finally, inspired by the recent work of our colleagues (Efremova et al., 2025), we sought to fuse P3/P4P3/P4-based agglomeration domains to the outward-facing C-terminus of the encapsulins in order to achieve control over the localization of the nanocages. However, given that the direct fusion of P3 and P4 with the encapsulins would likely impede the proper assembly of the nanocages and would not be susceptible to regulation by signaling cascades, we chose to adapt the SPOC design as developed by Professor R. Jerala (Fink et al., 2019). To learn more about how the final signaling cascades are envisioned for every approach, click here.

Tyrosinase engineering

As delineated above, tyrosinases constitute the core of our synthetic melanosomes. These proteins catalyze a one-enzyme reaction that leads to the synthesis of eumelanin, a dark pigment, from the naturally occurring AA tyrosine. As for melanin, tyrosinases occur in all domains of life, albeit with slight differences in their specific structures and functions. By definition, tyrosinases coordinate two copper ions, with each copper ion interacting with the three N_τ atoms of a histidine residue. These 6 residues are all comprised in the four α-helix bundle of the central domain, a structural element found in all tyrosinases. Additionally, several tyrosinases have been found to contain an N-terminal domain (NTD), typically a signal peptide, and a C-terminal domain (CTD) employed to regulate and modulate enzymatic activity (Pretzler and Rompel, 2024). We endeavored to capitalize on the extant structural knowledge to engineer our enzymes and regulate their activity within and outside of the synthetic compartments. Our main goal has been to reduce toxicity, both that resulting from uncontrolled melanin synthesis and that from excess copper in the cell. We selected promising tyrosinase candidates and strategies based on the existing literature, expert consultation with Prof. Pretzler and extensive structural modeling. To delve deeper into the respective chapters, click in the highlighted links or on the drop down chapter below.

Fig. 6 / Tyrosinase engineering Diagram of workflow to get tyrosinases with controllable activation.

Fig. 7 / Bacillus Megaterium Tyrosinase Green: Tyrosinase, Purple: Copper-coordinating histidnes, Orange: Copper

Split-Tyrosinases

Inspired by the split proteases that we encountered during our research on MESA, we hypothesized that creating a split tyrosinase could prevent melanin synthesis outside of the nanocages. We envisioned two separate tyrosinese fragments, both inactive as monomers, but with the ability to regain activity upon dimerization and reconstitution of the full protein, fused directly to the encapsulins. These constructs would remain inactive as monomers until the initiation of the assembly process of the nanocages. For this purpose we aimed to identify suitable tyrosinases that are characterized by a well-studied, simple structure devoid of complex CTDs, and most importantly, with possible splitting sites to disrupt the copper-coordinating central core domain.

Bacillus megaterium expresses a simple 35-kDa tyrosinase (BmTyr), the structure of which has been resolved at 2 Å by X-ray diffraction. The 297 AA enzyme has been demonstrated to dimerize as a result of hydrophobic interactions between AA Trp41 and Tyr267, as well as Arg37 and Asn270, while also being active as a monomer. Each monomer is composed of 8 α-helices, of which four assemble to form the active core with the six copper-coordinating residues. In the context of BmTyr, neither the C- nor the N-terminus possess significant functional domains (Sendovski et al., 2011).

We sought to rationally design our first split variant based on the crystal structure. Residue Pro85 is located in immediate proximity to Alpha-Helix 3 and separates three out of six functionally essential histidines. For the second split variant, we drew inspiration from the research conducted by P. Lee and his team. In their study, they identified a cp variant that retained tyrosinase function after the introduction of new termini at residues 201 and 202 (Lee et al., 2019). We hypothesized that if the Pro201 site, located before Alpha-Helix 7, was suited for the introduction of new termini in a cp variant, it might also be compatible with split versions. Subsequently, we encountered SPELL (Split Protein Reassembly by Ligand or Light), a computational tool capable of calculating the split energy of proteins, thereby predicting potential sites for protein splitting. Not only were the previously designed split sites (Pro85 and Pro201) confirmed, but an additional site (Lys157, situated prior to the a5 helix) that divides the first and last three histidine residues was also identified. We predicted the interaction of these split variants with copper ions with both AlphaFold 3 and Rosetta Commons (learn more about our model) and decided to test them in our wetlab segment (learn more about our results).

Caddie

This approach is predicated on the same rationale as for the split tyrosinases. Two distinct proteins, each inactive as a monomer, fused directly to the encapsulins. The process of nanocage assembly initiates reconstitution of a complex of both proteins, resulting in the formation of a functional complex. In this particular instance, the two proteins are not produced through the process of splitting an existing, functionally active tyrosinase. Rather, we identified two protein systems in nature and attempted to leverage them to circumvent the bottlenecks in our system.

Copper is a essential element for the optimal functionality of tyrosinase. However, elevated levels of copper are known to be toxic to cells. Caddie proteins possess two copper-binding sites and are capable of binding directly to tyrosinases, thereby facilitating the transport of copper into the active site. This interaction is especially crucial for proper functional tyrosinases when copper concentrations are relatively low, such as in physiological blood levels of approximately 0.1 μM (Matoba et al., 2011). We identified two distinct Streptomyces species, namely Streptomyces avermitilis and Streptomyces kathirae. Both tyrosinases represented compatible two-protein systems (SavMel and SkMel), after removal of the short exportation signals (Leu et al., 1992), that we decided to test within the context of our system.

Fig. 10 / SkMel with its caddie protein Green: Main Enzyme, Purple: Caddie Protein

Lid

Another intriguing phenomenon observed in nature is the so-called lid domain, which hinders substrate from accessing the catalytic center of the enzyme, thereby impeding its activity. In contrast to the predominantly helical central domain, this CTD is usually composed of a beta-barrel like structure and a flexible linker that connects it to the catalytic center. The regulatory mechanism in the cell is contingent upon the precise structural characteristic of the lid domain and its linker. In general, lids can be classified as either self-regulatory or actively regulated. Self-regulatory lids can undergo autoproteolysis or switch between open and closed conformations in response to environmental cues, such as pH and temperature. Actively regulated lids are modulated by additional proteins, such as proteases. While the idea of modulating tyrosinase activity through proteases appeared compelling, hurdles emerged concerning the identification of suitable candidates, primarily with regard to the stability of the active state, which is generally prone to degradation (de Almeida Santos et al., 2024; Son et al., 2018).

Hahella expresses not one, but two different Tyrosinases with lid domains (HcTyr1 and HcTyr2). HcTyr2 demonstrates a propensity for degradation and agglomeration, a significant drawback in our system. In contrast HcTyr1 exhibits a higher degree of stability and is less susceptible to degradation upon autoproteolysis of the lid domain (de Almeida Santos et al., 2024). We conducted a comprehensive review of the extant literature and compared the protein sequences of various lid-domain-containing tyrosinases to identify the segments responsible for autoproteolysis. We substituted these residues with a TEV cleavage site and confirmed our designs by modeling before proceeding with experimental validation of this specific part. Furthermore, we consulted with Dr. Pretzler from the institute of biophysical chemistry of the universität wien. Following his advice we implemented the same pipeline for the tyrosinase of Verrucomicrobium spinosum (VsTyr), which resulted in a second TEV-proteases activable tyrosinase design (Fekry et al., 2023).

Fig. 11 / HcTyr1 The 3D Structure of HcTyr1

Functionally active tyrosinases and mutants

We aimed to test these engineereing approaches on the previously mentioned tyrosinases but conceived them as being applicable to other enzymes as well. We selected wild-type BmTyr and several variants that we encountered during our research process in hope for increased reaction rates. These included the aforementioned cp-BmTyr (Lee et al., 2019), and the three mutants BmTyr E195S A221V from Kang et al. (2024), BmTyr R209S M215E V218M and BmTyr G43R M61H F197W Q214D V217A A232C from Liu et al. (2024). Moreover we found more efficient mutants of SavMelC1 (Y92F) and SavMelC2 (I42Y) that we decided to analyze (Lee et al., 2015). Finally, we noted that the tyrosinase from Laceyella sacchari (LsTyr) exhibited unusually high kcat values for the L-Dopa reaction (according to Dolashki et al. (2015)), thus prompting our decision to include it in our set of tested tyrosinases.

We attempted expression of the selected tyrosinases by means of cell-free expression, expression in E. coli and in HEK293T, in order to assay the activity of the engineered enzymes. The results of our work can be found here!

Biomarker Targets Research

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What is this target?

Progesterone is a steroid hormone primarily produced by the corpus luteum during the luteal phase of the menstrual cycle and by the placenta during pregnancy (Strauss, 1986). It is a key regulator of the menstrual cycle and pregnancy maintenance, with levels varying from 0.1-0.89 ng/mL in follicular phase, to 7-25 ng/mL in luteal phase (University of Rochester Medical Center, 2025). For its role in the menstrual cycle, progesterone alsoo serves as a biomarker for fertility tracking (ovulation confirmed at ≥3 ng/mL by Leiva et al. (2015)) and ectopic pregnancy detection (levels < 5 ng/mL during pregnancy indicate high risk with 88% sensitivity according to (Murray et al., 2005)).

What makes this target interesting?

Evidence shows that small, uncharged molecules like progesterone readily diffuse from blood to interstitial fluid with nearly identical concentrations, meaning tattoo cells would have direct access to physiologically relevant hormone levels, as an interstitial fluid-based wearable biosensors described by Wu et al. (2024). A 10-25x fold difference between follicular phase (<1 ng/mL) and post-ovulation luteal phase (7-25 ng/mL) provides an ideal bimodal expression pattern and a strong signal for color change visualization, perfect for binary “fertile/not fertile” readout (University of Rochester Medical Center, 2025). This graph, adapted from Häggström (2014), provides an excellent overview of this bimodal pattern:

adapted from Häggström (2014) and licensed under CC0 1.0.

Chen et al. (2020) showed that at 314.46 Da, the analyte progesterone can efficiently penetrate hydrogel matrices and cell membranes, ensuring fast response times for pigmentation change. Since progesterone is membrane-permeable, it enables the inclusion of intracellular MESA, which we have used in our software validation design.

Thus, the specific monitoring purpose can be targeted by setting activation limits for the tattoo accordinglyprogesterone provides an excellent target for a multitude of purposes which can be targeted by setting activation thresholds for the tattoo accordingly. As described previously, a 10-25x fold difference between different cycle phases enables a fertility readout and additionally, very low progesterone levels during pregnancy indicate ectopic pregnancies, which could be detected and treated early enough via this method.

Such distinguishable spikes in the concentration are also well-suited for the detection using InkSight. Unlike single blood tests that only provide snapshots in time, biosensor tattoos can enable continous tracking. Existing continuous platforms already claim 46% improved IVF success rates (OOVA Inc., 2025), and Stovall et al. (1989) showed that it could reduce ectopic pregnancy ruptures by 79.2% to 38.8%.

Structure and Modelling

Progesterone is composed of a four-ring structure (A-B-C-D rings) characteristic for steroid hormones, with ketone groups at C3 and C20 positions, providing specific recognition sites for engineered receptors (ChemicalBook, 2025). Its high lipophilicity (LogP: 3.87) causes poor water solubility (~16.8 μg/mL), aiding in membrane permeability for cellular uptake. Unlike other targets, progesterone is a small organic molecule, providing advantages for stability (no denaturation) and consistent diffusion through the cellular matrix.

Using a crystal structure (Pacesa et al., 2024) of progesterone-bound DB3 Fab in complex with computationally designed DBPro1156_2 protein binder (Marchand et al., 2025), we were able to successfully create a MESA receptor for progesterone. We are currently evaluating laboratory testing of this soluble MESA Construct.

Limitations

The first limitation concerns the specificity and tunability of the biosensor. Despite its advantages, several potential issues remain when using progesterone to track the female menstrual cycle and detect ectopic pregnancies. McKay (2023) reported that progesterone levels exhibit a coefficient of variation of 0.61–0.66 between cycles, which may require personalized calibration of tattoo color thresholds. Additionally, extensive trials are needed to determine the precise hormone concentrations that trigger contrast switching in the tattoo. While diffusion and equilibration between interstitial fluid (ISF) and blood are generally reliable, some studies, including Cicinelli et al. (2009), suggest there can be up to a twofold difference in progesterone levels, and complete data across the entire menstrual cycle are currently lacking. Further measurements and trials are therefore required to establish accurate thresholds.

Another major challenge is the timing of the biosensor response, as studies of intravenously injected progesterone report half-lives ranging from 3 to 90 minutes (Aufrère and Benson, 1976). This indicates that the tattoo may need to respond rapidly to concentration changes, though this is likely less critical under natural, continuous hormone production compared to artificial injections.

Finally, detection specificity requires careful attention. Current immunoassays show significant analytical bias versus LC-MS/MS due to structural similarities with cortisol and testosterone (Debeljak et al., 2020). Engineered cells must therefore achieve high specificity to avoid cross-reactivity and potential false-positive readings.

Conclusion and Outlook

In summary, research indicates that progesterone is detectable in the interstitial fluid and a monitoring platform like InkSIght may offer a less invasive, but most importantly continous alternative to blood testing. The InkSight system, implementing a MESA receptor presents promising opportunities for advanced biosensing applications. Establsihing individual baseline levels may enable more precise monitoring of progesterone. After talking with an expert in progesterone detection as well as artists, we believe that through tracking of the menstrual cycle, progesterone monitoring can support fertility tracking, assist in fertility treatments, and help manage progestin supplement intake.

What is this target?

The troponin complex is a critical regulatory protein bound to the actin filaments within cardiac and skeletal muscle cells, playing an integral role in the contractile process. Its function is governed by a precise molecular interplay: according to Han et al. (2025), troponin I (TnI) acts as an inhibitor, preventing muscular contraction in the absence of calcium. Initiation of contraction is triggered when calcium ions bind to Troponin C (TnC), inducing a conformational shift in the entire complex. Li and Hwang (2015) found that this structural change displaces Troponin I, lifting its inhibitory effect and allowing contraction to proceed.

The diagnostic power of Troponin I stems from its exclusive expression in cardiomyocytes, as confirmed by single-cell RNA sequencing data (The Human Protein Atlas, n.d.). This tissue specificity makes its presence in blood plasma a highly sensitive and specific indicator of cardiac cell damage. While elevated TnI levels are a cornerstone for diagnosing acute myocardial infarction (Potter et al., 2022), (van Beek et al., 2016), emerging research underscores its significant value as a potent prognostic indicator, providing critical insights into future cardiovascular risk beyond merely identifying past injury.

What makes this target interesting?

The development of a highly sensitive and accessible Troponin I (TnI) biosensor is poised to address a profound and escalating global health challenge. Cardiovascular disease (CVD) persists as a leading cause of mortality worldwide [WHO_CVD_FactSheet_2024], creating a vast and growing population of individuals living with elevated risk. The strategic value of this diagnostic tool is underscored by the broad spectrum of stakeholders it stands to benefit.

The healthcare system with resource-limited settings can profit in the delivery of cardiac care in underserved regions. By facilitating point-of-care diagnosis without reliance on sophisticated laboratory infrastructure or regular health check-ups, it can bridge critical gaps in healthcare access and enable timely triage and treatment.

The most significant impact will be felt by patients, especially the substantial number with a history of cardiac events who face a persistently high risk of recurrence. Approximately one in five heart attacks is recurrent (Tsao et al., 2023). An accessible monitoring tool fosters patient empowerment, enables patient-centric care models, and ultimately paves the way for improved long-term health outcomes. We spoke with two people affected. Here's what they had to say.

Troponin I presents an optimal target for diagnostic development due to a compelling combination of molecular and practical advantages:

• Favorable biophysical properties: With a molecular weight of 23.9 kDa (“Troponin I – an overview,” n.d.), TnI is an ideal size for detection using MESA technology and ensures efficient diffusion through hydrogel matrices, which is critical for detection performance.
• Well-defined epitopes: The protein is structurally well-characterized, with existing antibody-binding structures available (e.g., PDB 4P48), facilitating the design of high-affinity capture ligands.
• Optimal clinical range: TnI is present in blood plasma at a measurable baseline concentration 1.1nl/ml (The Human Protein Atlas, n.d.) in healthy individuals. The concentration rises dramatically upon injury. This wide dynamic range ensures a reliable and quantifiable output signal.

Structure and Modelling

Structurally, TnI forms an α-helical coiled-coil with TnT, creating a rigid, asymmetric segment known as the IT arm that spans the actin filament and anchors tropomyosin (Takeda, Satoru and Yamashita, Atsuko and Maeda, Kenji and Maeda, Yuichiro, 2003). Overall, TnI acts as the inhibitory subunit that converts Ca²⁺-induced structural changes in troponin into mechanical regulation of muscle contraction.
Using BindCraft (Martin Pacesa et al., 2024) to create one de-novo binder as well as a naturally-occuring one on differnet epitopes, these binding sequences were reported to have good affinity metrics and were sterically configured to offer a possible dimerization.

Limitations

While the potential for false negatives is considered low, its impact would be significant, necessitating extensive confirmatory testing. A more probable challenge is the occurrence of false positives. These are primarily attributed to:

• the inherent binding characteristics of the receptor, and
• the complication of monitoring patients with pre-existing elevated Troponin I (TnI) levels due to stress or physical exercise

Conclusion and Outlook

In conclusion, Troponin I (TnI) stands out as a highly promising target for next generation cardiac diagnosis due to its combination of exclusive expression in cardiomyocytes, sharp elevation during cardiac injury and biophysical copatibility with biosensor platforms like MESA. Its exclusive expression in cardiomyocytes, sharp elevation during cardiac injury, and strong prognosis value make it an ideal candidate for both acute and long-term cardiovascular monitoring. The development of a sensitive, accessible TnI biosensor holds significant potential to transform cardiac care, particularly in underserved regions where rapid, point-of-care diagnostics could dramatically improving circuit response times. With continued development, a TnI-based diagnostic tool could bridge critical gaps in cardiac care and empower patients with real-time insight into their cardiovascular health.

What is this target?

Progestagen-Associated Endometrial Protein (PAEP), more commonly known as Glycodelin or PP14 (Placental Protein 14), is a multifunctional glycoprotein that has gained significant interest in oncology research (Uhlén et al., 2015) for its potent role in immune modulation and hormone response. Its primary physiological function is established in the female reproductive system, where it downregulates the maternal immune response to protect the fetus during pregnancy (Bersinger et al., 2009). However, this same immunosuppressive mechanism is co-opted by various cancers, making PAEP a promising biomarker for therapy monitoring and tracking tumor progression (Richtmann et al., 2024).

In tumor microenvironment, PAEP is significantly overexpressed and contributes directly to immune resistance. By suppressing the activity of natural killer (NK) cells and T-cells, it enables tumors to evade host immune surveillance. This role is of critical clinical importance in the era of immunotherapy. For instance, elevated serum glycodelin levels prior to treatment have been correlated with worse outcomes in female cancer patients treated with PD-(L)1 inhibitors, monoclonal antibodies targeting the PD-1/PD-L1 axis to prevent tumor-induced immune evasion (Schneider et al., 2015). Consequently, monitoring dynamic changes in PAEP levels during therapy could provide a vital window into treatment efficacy. A decrease may indicate the tumor is becoming less immunosuppressive, a positive response, while a sustained or rising level could serve as an early warning of adaptive immune escape and emerging treatment resistance, thereby guiding timely adjustments to immunotherapy strategies.

What makes this target interesting?

While PAEP is most studied in cancers of the reproductive system, its relevance is not gender-restricted. It is also expressed at significant levels in male reproductive tissues, the lungs, and the prostate. For example, research has confirmed that lung tumors produce PAEP mRNA, leveraging its immunosuppressive function to their advantage (Authors, 2025). This broad expression profile underscores its potential utility as a cancer biomarker. Therefore, PAEP represents a compelling therapeutic indicator, whose monitoring could personalize and enhance the effectiveness of modern immunotherapies across a range of malignancies(Bersinger et al., 2009).
For instance, in non-small cell lung cancer (NSCLC), PAEP is not only expressed but actively secreted, with mRNA overexpressed in approximately 80% of tumors. Its knockdown directly downegulates key immune checkpoints like PD-L1, explaining its active role in immune evasion (Richtmann et al., 2024).

Crucially, serum Glycodelin levels have been demonstrated to correlate with patient response to treatment with a broad expression profile from male reproductive tissues and the prostate to the lungs (Richtmann et al., 2024), (Schneider et al., 2015), (Bersinger et al., 2009).In healthy individuals, baseline serum levels are very low, around 110 ng/mL [uhlen_tissue-based_2015]. However, these levels can become dramatically elevated in pathological states. For instance, pre-surgical serum levels have been reported with a median of 8.9 ng/mL in those cancer patients with high tumor PAEP expression. Values can exceed 228 ng/mL, significantly higher than in non-cancer controls. Elevated levels are also found in the tumor interstitial fluid, highlighting its active local secretion(Schneider et al., 2018, 2015).

Structure and Modelling

The glycoprotein has a molecular weight that typically ranges from approximately 28 to 35 kDa (Uhlén et al., 2015). This variation is primarily due to its significant and heterogeneous glycosylation, meaning the protein core is decorated with diverse sugar chains. Using BindCraft (Martin Pacesa et al., 2024) to create de-novo binders on differnet epitopes, these binding sequences were reported to have good affinity metrics and were sterically configured to offer a possible dimerization.

Limitations

The regulatory mechanisms and biological functions of glycodelin are highly complex and significantly complicate its potential leverage as a biomarker. First, physiologic variability is pronounced. In premenopausal women, serum glycodelin rises late in the luteal phase and is tightly linked to progesterone, making single time-point values difficult to interpret without cycle staging (Bersinger et al., 2009). Secondly, they lack specificity of disease. Elevations occur in several benign gynecologic conditions, especially endometriosis (Kocbek et al., 2013). Third, glycodelin exists in multiple glycoforms with distinct immunomodulatory effects, so measured concentrations may not reflect the bioactive species relevant to antitumor immunity (Lee et al., 2009).

Conclusion and Outlook

Current evidence positions PAEP as a biologically grounded but still experimentally maturing biomarker in oncology. Its detectable secretion by tumors and the resulting serum presence in several malignancies, particularly non-small cell lung cancer (NSCLC)(Schneider et al., 2015), underscore its potential for therapy monitoring and disease surveillance. However, translating this potential into clinical routine will require a nuanced understanding of its physiological variability and context-dependent regulation.
Glycodelin’s strong hormonal sensitivity and dual role in reproduction complicate interpretation because fluctuations in serum levels can originate from non-malignant gynecologic sources, notably endometriosis, where elevated PAEP concentrations are observed both locally and systemically. While such overlap may limit its specificity as an isolated cancer marker, it also highlights an opportunity—the same biology that confounds oncologic interpretation could be harnessed diagnostically to monitor endometriosis progression and therapeutic response.

What is this target?

Erythropoietin (EPO) is a glycoprotein hormone primarily synthesized in the kidneys under the regulation of hypoxia-inducible transcription factors (HIFs). It is essential for the survival and proliferation of erythroid progenitors in the bone marrow, making it a crucial regulator of red blood cell (RBC) production.
EPO production occurs mainly in peritubular fibroblasts of the renal cortex, where tissue oxygen levels (pO₂) act as the key regulatory signal. This location is particularly well-suited for oxygen-sensitive EPO synthesis, as renal cortical pO₂ remains relatively stable despite changes in blood flow, allowing the kidneys to reliably monitor blood oxygen content determined by hemoglobin (Hb) concentration, arterial oxygen pressure, and Hb–O₂ affinity.
Although the liver serves as the primary source of EPO during fetal development, smaller amounts are still produced in adults in the liver, spleen, bone marrow, lungs, and brain. (Jelkmann, 2013) , (Kettelhack et al., 1994) , (Yin and Noguchi, 2025)

What makes this target interesting?

EPOs’ deficiency is the primary cause of anemia in chronic kidney disease (CKD) (Jelkmann, 2013). Not uncommonly, cancer patients experience anemia post-treatment-induced bone marrow suppression, which leads to a decrease in red blood cell (RBC) production. Thus, in anemia management EPO serves as a critical therapeutic biomarker (Yin and Noguchi, 2025).
The recombinant human EPO (rhEPO, epoetin) has become a cornerstone treatment for both CKD-related anemia and anemia in cancer patients undergoing chemotherapy (Jelkmann, 2013). The fundamental goal of EPO therapy is to stimulate the bone marrow to increase RBC production, thereby alleviating anemia symptoms and, most importantly, reducing the need for blood transfusions and their associated risks (Yin and Noguchi, 2025).
Recent advancements have expanded the therapeutic use of EPO. Several patent expirations of rhEPO have enabled the production of biosimilar agents. In addition, improved analogs such as darbepoetin alfa and methoxy polyethylene glycol-epoetin beta have been developed to offer prolonged circulation half-lives (Schoener B, 2025). Novel erythropoiesis-stimulating agents are also under clinical investigation, including direct stimulators like EPO-mimetic peptides and activin A-binding proteins, as well as indirect agents such as HIF stabilizers that promote endogenous EPO synthesis (Jelkmann, 2013).
However, administration of rhEPO or its analogs carries risks, including an elevated incidence of thromboembolic events (Schoener B, 2025).

Current monitoring methods

Monitoring EPO levels is essential for establishing a baseline and guiding treatment. In healthy individuals, normal circulating EPO concentrations typically range from 4 to 26 mU/mL (Kadali and Turley Jr PA-C, 2025) , (Jelkmann, 2013). It is important to note that dosage regimens are highly variable and must be individualized. To achieve a strong, targeted spike in RBC production from the bone marrow to overwhelm the suppressed state of the bone marrow and forcefully stimulate erythropoiesis, clinicians administer very large, supra-physiological concentrations of recombinant EPO. For the chemotherapy-related anemia it recommended to start the therapy with 40,000 units subcutaneously once weekly, 3 times per week (Schoener B, 2025).
Currently patients undergo mandatory daily or weekly blood tests, but even there not the concentration of the therauputic is tested, but corresponding hemoglobin levels.(Schoener B, 2025) The tests aim to track hemoglobin and hematocrit levels with high resolution, ensuring the dosage is titrated perfectly to avoid excessive amounts. In clinical settings, enzyme-linked immuno-sorbent assays (ELISAs)are commonly used to measure EPO immunoreactivity units (U) (Jelkmann, 2013).

Structure and Modelling

Erythropoietin is a fully glycosylated, biologically active glycoprotein hormone. Its biological activity is intrinsically linked to its complex molecular structure. The active hormone consists of a 165-amino acid polypeptide backbone that is heavily modified by carbohydrate chains, which account for approximately 40% of its total molecular mass. This extensive glycosylation is essential for the hormone’s biological activity, stability, and solubility, resulting in a final molecular weight of roughly 30-34 kDa (Jelkmann, 2013). Its size allows the molecule to easily travel into the interstitial fluid, where InkSight is localized.
A key pharmacological characteristic of EPO is its relatively short half-life. The pharmakokinetics of the EPO therapy shows different results based on the modification. Epoetin alpha has a relatively short plasma half-life: after intravenous administration it’s approximately 6 hours, while via subcutaneous injection it can extend to around 24 hours. Darbepoetin alfa, thanks to additional glycosylation, shows a longer terminal half-life around 48.4 h after subcutaneous administration in adults (Czock and Keller, 2021). This brief duration is a primary reason why patients require frequent dosing to maintain the necessary stimulus for red blood cell production. (Schoener B, 2025)

Limitations

Even with its diverse portfolio of therapy strategies, the therapeutic use of EPO still creates a clinical paradox: the same mechanism that alleviates anemia also introduces substantial risks:

• The artificially induced surge in RBC production directly increases the likelihood of life-threatening thrombotic events (blood clots) and may adversely affect tumor outcomes if hemoglobin levels rise too rapidly.
• Additionally, the increase in blood viscosity associated with elevated hemoglobin levels has been linked to cases of severe hypertension (Jelkmann, 2013).

Analytically, EPO‘s low basal concentration in the pico-molar range demands extreme assay sensitivity, while its structural heterogeneity from glycosylation isoforms and the presence of recombinant variants complicate accurate quantification. Furthermore, the complex serum matrix creates a high risk of cross-reactivity and non-specific binding, potentially leading to misleading results (Jelkmann, 2013). Overcoming these analytical limitations is therefore not just a technical exercise, but a prerequisite for enabling the safe and effective application of EPO-targeting therapies.
Another major challenge is factoring in the influence of additional factors such as infections or individual physiological differences, which can alter red blood cell counts and consequently affect EPO levels (Kadali and Turley Jr PA-C, 2025).

Conclusion and Outlook

Implementing InkSight as a detection system for EPO could enable less invasive and more continuous monitoring, reducing patient stress and the frequency of blood sampling, protecting from the negative concequences of the high EPO concentrations. The modularity of a MESA receptor would allow to conveniently change the binding site, adapting to a variety of EPO-derived therapeutics. For realistic implementation, however, it will be important to control confounding factors such as variable tissue oxygen tension (pO₂) to increase measurement precision. As research in EPO biology and therapy continues to advance, such innovations could bridge the gap between therapeutic efficacy and real-time monitoring, making EPO management safer and more efficient.

What is the target

LL-37 is the sole human cathelicidin antimicrobial peptide (CAMP). It plays a dual role as both a direct antimicrobial agent and a potent modulator of the immune response.

It exhibits broad-spectrum antibacterial activity, effectively targeting both Gram-positive and Gram-negative bacteria.(Morizane et al., 2012) It is endogenously expressed in various human cells, including keratinocytes, neutrophils, and mast cells, and is consequently found at high concentrations in inflamed skin and blister fluids. Beyond its direct antimicrobial role, LL-37 is a recognized modulator of inflammatory responses and is believed to play a key part in wound healing processes. Its clinical relevance is further underscored by its demonstrated efficacy against specific respiratory pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. (Reczyńska-Kolman et al., 2025) (Majewski et al., 2018)

It is a well-established biomarker in inflammatory skin pathologies, most notably psoriasis and rosacea. In psoriasis, damaged keratinocytes in the epidermis and dermis release LL-37, which then acts as a pro-inflammatory mediator, fueling the disease cycle.(Geng et al., 2024)

Beyond the skin, chronic inflammation leads to elevated systemic levels of LL-37. Emerging research reveals a direct link to an increased risk of cardiovascular disease (CVD). LL-37 can bind to low-density lipoprotein (LDL), forming a complex that is more readily internalized by macrophages via receptors like LDLR and CD36. This process accelerates foam cell formation, a critical step in atherosclerosis. This mechanism is unique to human and some primate cathelicidins and provides a molecular explanation for the elevated CVD risk observed in patients with chronic inflammatory disorders like psoriasis.(Reczyńska-Kolman et al., 2025)(Ong et al., 2002)

While systemic blood concentrations can vary, a consistent trend shows LL-37 levels are elevated in patients with active disease. Histological analyses confirm that psoriatic lesions exhibit significantly more intense staining for LL-37 compared to healthy skin. This overexpression is a hallmark of the condition.(Morizane et al., 2012)

How is the target interesting

The strong correlation between LL-37 levels and disease activity is well-documented. Clinical insight suggests that monitoring its dynamics could have predictive value, potentially forecasting disease flare-ups weeks in advance. This makes it a highly promising target for a diagnostic sensor aimed at tracking disease progression and therapy response directly within the skin.

Biologically, it is naturally present in the dermal compartment and its concentration in interstitial fluid (ISF) shows a strongly bimodal distribution between healthy and inflamed skin, providing a robust diagnostic signal. Clinically, it functions as both a predictive marker and a tool for therapeutic monitoring, allowing patients to optimize medication use based on objective data, as suggested by clinical partners. Technically, its small size (37 amino acids) ensures facile penetration into the hydrogel matrix of the sensor, enabling efficient detection.(Yang et al., 2020) , (Reinholz et al., 2012)

Structure and Modelling

The human cathelicidin protein consists of an N-terminal cathelin domain and the C-terminal LL-37 peptide. This active LL-37 fragment is released by proteolytic cleavage, forming a mature peptide of 37 amino acids characterized by two N-terminal leucines and a net charge of +6. Its structure has been extensively studied, revealing significant conformational plasticity dependent on the environment. Early NMR analyses in denaturing SDS micelles revealed a monomeric, kinked helix. In contrast, more recent crystallographic data in aqueous environments identified an anti-parallel dimer. This dimer undergoes substantial structural rearrangement upon interacting with detergent micelles like DPC or LDAO, which serve as membrane mimetics. High-resolution structures from these conditions have defined stable lipid-binding sites, primarily involving key aromatic residues. These same interaction sites have been confirmed by NMR in DPC, mapping onto a consistent tripartite structure that is believed to reflect its mode of action on biological membranes in vivo.(Sancho-Vaello et al., 2020)

Limitations

Detecting LL-37 in the interstitial fluid (ISF) presented several challenges. The biomarker is typically present at very low concentrations in biological fluids like ISF, making it’s detection difficult (Ridyard and Overhage, 2021). Additionally, proteolytic degradation caused by the proteases in bodily fluids can reduce the amount of intact LL-37, further complicating accurate quantification.(Grönberg et al., 2011) Furthermore, LL-37 may undergo citrullination - a process where the amino acid argine is converted to citrulline withing the protein (Coenen et al., 2007). This modification can alter LL-37’s function and promote immune response, potentially changing its properties and making detection even more challenging (Al-Adwani et al., 2020).
Lastly, inflammation can be caused simply by external factors such as local skin irritation or infection potentially makingdisease-driven fluctuations in biomarker levels.

Conclusion and Outlook

The inspiration for investigating LL-37 as a diagnostic target originated from our discussions with Dr. med. Sebastian Volc, a renowned dermatologist and expert in biosensor technology. Given the well-established correlation between LL-37 expression and disease activity, tracking its dynamics could provide valuable predictive insight, helping to identify inflammatory flare-ups earlier and simplifying therapeutic decision-making.

While our research acknowledged the inherent challenges, including LL-37’s natural expression in the dermal compartment and its strongly bimodal concentration distribution between healthy and inflamed skin, these very features give benefit to its diagnostic potential. LL-37’s specificity of expression and sensitivity to inflammation make it a compelling candidate for continued InkSight development. With further optimization, LL-37 monitoring could contribute to a continuous assessment of inflammatory skin conditions and enhance personalized dermatological care.

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Mechanisms for contrast change

Subsequent to the selection of all components of the system, the elements were assembled in a functional manner. In general, color changes at the cellular level can be achieved through several strategies: production, degradation, modification or relocation of chromatophores. While the selective degradation or controlled modification of encapsulated pigments proved to be a daunting challange, we have successfully developed strategies based on prodution and relocation of pigments. The aforementioned synthesis of melanin in mammals depends on intrinsic and extrinsic factors (e.g., sunlight in tanning), making it an excellent example of color change through chormatophore production (Gilchrest, 2011; Maranduca et al., 2019; Raposo and Marks, 2007). In such systems, the amount of pigment produced and available in a cell mainly regulates contrast, resulting in relatively slow tanning process (Gur et al., 2024). On the other hand, rapid color change can be achieved by relocating chromatophores, as seen in Tilapia mossambica, also known as the African cichlid. Their melanophores exist in two states. Somewhat conterintuitively, the pigment is concentrated at the cell center in the light state, which reduces the amount of absorbed light. In the dark state, their pigments are distributed across the cytosol to maximize light absorbtion. Again, this process is mediated by complex cytoskeletal transport processes that we cannot replicate. Instead, we tried to mimic them to achieve the same effect (Haimo and Thaler, 1994). The following section will provide a detailed description of the configurations that enable selective color / contrast changes upon ligand binding to the receptor.

Expression

This mechanism is arguably the simplest and slowest, relying on gene expression to achieve a contrast change. It involves a MESA receptor with a protease, ideally a split protease, and a transcription factor that directly activates the expression of encapsulin-tyrosinase fusion proteins. In the case of split-tyrosinase fusions, these proteins do not produce melanin until the spontaneous assembly process is initiated. Over time, the amount of melanin in the assembled nanocages will rise, leading to increased light absorption and a darker appearance. This pigmentation would persist until the degradation of the synthetic organelles, which, according to our estimation based on turnover rates of peroxisomes, could occur within a range of several hours to days, contingent on the size and exact composition of the nanocages (Huybrechts et al., 2009). Due to the reaction times of this mechanism, its use for rapid detection of targets, such as required for troponin, is not feasible. However it could be utilized for targets with relatively slow kinteics such as progesterone. In this particular instace, the elevation of progesterone levels, which typically occurs immediately preceding ovulation, would result in the darkening of the tattoo. Conversely, during the inital half of the menstrual cycle, it would remain in its light (invisible) state.

Assembly

In an effort to enhance reaction times, we formulated a second strategy, that does not rely on regulation of protein expression levels. Here, both encapsulins fused to sterically hindering Lanthanide-Binding Tags (LBT) and lid-tyrosinases with terminal encapsulation signals are constutively expressed. The inhibitory domains, which are cleavable through included TEV cleavage sites in the respective linker regions, will lead to an inactive state of the respective proteins until receptor activation. The receptor entails IED linker containing TEV cleavage sites and a split TEV protease. Upon binding of the target, the receptor dimerizes, which leads to the reconstitution and activation of the protease. The reconstituted enzyme cleaves the intracellular linker, thereby releasing itself from the membrane. Subsequently, the activation of both encapsulins and tyrosinases is achieved through the removal of LBTs and lid-domains, respecively.

In this strategy, transcription of the required proteins does not begin upon ligand binding to the receptor. Rather, at the time of detection, all functional components are already available in the cytosol and only necessitate activation to initiate pigment synthesis. Therefore, gain in contrast is predominatly contingent upon the efficacy of the selected tyrosinase. For this reason we developed a mathematic model to calculate the syntheis rates of different tyrosinases. Furthermore, we attempted to caclulate the requesite number of cells / hydrogel beads necessary to generate visible contrast. Check out our progress here. Generally, we hypothesized this mechanism to react more quickly to targets, thus enabling its deployment in the monitoring of e.g. EPO therpies. In this context, the tattoo will be colorless until the detection of an elevated dosage of EPO, which leads to activation of the encapsulins and tyrosinases and to generation of visible contrast.

Agglomeration

Finally, the agglomeration mechanism represents our most rapid color changing strategy. For this concept, active encapsulins, fused to either active tyrosinases or, ideally, split tyrosinases are constitutively expressed. The continuous assembly of active nanocages, which contain functional melanin-synthesizing enzymes, will result in a generally dark appearance of the tattoo until detection of a ligand. The C-terminus of the encapsulin is equipped with a TEV protease activable agglomeration domain, such as SPOC. The receptor design is consistent with the one previously mentioned for assembly, consisting of a split TEV protease that is capable of cleaving itself off the membrane upon activation of MESA. Subsequently, the active protease will cleave the SPOC linker, which will result in the dissociation of the inhibiting domain. This process is designed to prompt the fast agglomeration of the nanocages. In a manner analogous to the process described for Tilapia mossambica, the concentration of the synthetic melanosomes at a singular focal point results in diminished light absorption and thus in a depigmentation of the tattoo. This rapid decolouration mechanism can find application in the detection of troponin, a marker which necessitates immediate intervention in cases of elevated levels.

Outlook

After having developed a proof of concept, several ideas emerged for how the project could evolve beyond the iGEM framework. Our intention from the beginning was to create something that could prove legitate even outside the competition. The following outlook summarizes the directions that, through expert discussions, survey results, and internal reflection, appear most promising for future development.

Pigments

For this proof of concept, we chose eumelanin as our pigment primarily for reasons of feasibility within the limited project timeframe. Its formation requires only a single enzyme and naturally results in supramolecular structures that are readily confined within encapsulin nanocages—making it both practical and technically robust for initial implementation.

Looking ahead, however, we see the necessity of expanding beyond eumelanin. Developing genetic circuits capable of producing a broader palette of pigments could allow for multi-colored, high-contrast biosensors. Most importantly, by replacing the current startegy of pigmentation with one where contrast and visibility are no longer dependent on skin color, could adress the major challenge of inclusivity in this diagnostic design. Simultaneously, it may open the door to multiplexed sensing—where distinct pigments could report on different biomarkers simultaneously.

[Betalains] are pigments mostly found in plants where they are responsible for the vibrant color in patels and give beetroot its distinctive color. They are divided into betacyanins (red–violet; λ^max ≈ 535–550 nm) and betaxanthins (yellow–orange; λ^max ≈ 461–485 nm).

Just like in the synthesis pathway of melanin, the precurser molecule for betalains is L-tyrosine. From L-tyrosine, a short 3-step pathway yields color: (1) tyrosine is converted to L-DOPA and L-dopamine (in plants via CYP76AD P450s), (2) DOPA-4,5-dioxygenase (DOD/DOD1) cleaves L-DOPA to betalamic acid (chromophore; ~405–410 nm), (3) non-enzymatic condensation of betalamic acid with amino acids produces betaxanthins (yellow), or with cyclo-DOPA/cDOPA-5-O-glucoside produces betacyanins (red–violet).

In engineered hosts like yeast and plants, a transgene expression cassette with 2-3 genes, namely CYP76AD, DOD and optionally a glycosyl transferase has successfully been inroduced to ensure visible pigment formation (Khan and Polturak, 2025).

Another, more advanced option for pigments are reflectins. Just as the concept of a cell-based biosensor tattoo was originally inspired by cuttlefish, this organism offers another intriguing feature that could be leveraged. Among the cuttlefish’s specialized skin cells are leucophores, broadband reflectors that appear white because they diffusely scatter all visible wavelengths. Leucophores are densely packed with disordered, micron-scale scatterers called leucosomes, which act as broadband Mie scatterers. Their random spatial arrangement ensures that light is scattered in all directions and across the visible spectrum, resulting in a bright, matte white appearance.

Want to learn more about Reflectins?

Many of the internal photonic architectures of cephalopod skin, including leucosomes, are largely composed of reflectins. These proteins feature highly conserved motifs separated by aromatic and charged linker regions, a sequence composition that underlies their remarkable ability to self-assemble into diverse nanostructures (including spheroids, fibers, hexagonal platelets, and thin films) and to exhibit exceptionally high refractive indices. For example, spheroidal leucosomes in cuttlefish leucophores have a refractive index of roughly 1.51, considerably higher than that of typical cytoplasm (≈ 1.35). This large index contrast is what enables strong scattering and broadband reflection.

Research into reproducing this phenomenon in mammalian cells is still in its early stages. In a landmark study, Chatterjee et al. (2020) expressed reflectin A1 (RfA1) in HEK cells and demonstrated that the protein self-assembled into spheroidal, leucosome-like aggregates throughout the cytoplasm. These aggregates measurably increased the average cytoplasmic refractive index, and spectrophotometric analysis revealed a twofold increase in diffuse transmittance and reflectance relative to control cells, confirming enhanced light scattering. Importantly, reflectin expression did not significantly reduce cell viability. Nevertheless, the engineered cells did not yet appear bright white to the naked eye. The measured diffuse reflectance was only about 2 %, far below the 30–40 % reflectance typical of natural leucophores. Macroscopically, the cells appeared slightly more opaque or cloudy, rather than forming a clearly visible white patch. Thus, while this work provides an important proof-of-principle that reflectin-based scattering is possible in human cells, the technology is still far from producing the intense whiteness required for practical applications such as a visually distinct biosensor tattoo.

Another fascinating angle for achieving a visible output that is more inclusive is the use of guanine platelets as a structural color element. Guanine is a purine nucleobase that can be functionalized for optical formation of broadband reflectors in fish scales, insect cuticles, and scallop eyes. Its exceptional refractive index (~1.83) and ability to assemble into thin, highly ordered platelets make it one of the most efficient biological materials for light reflection. In organisms, these platelets are arranged into stacks of multiple layers, creating mirrors and diffuse reflectors with up to 30–40% reflectance.

Want to learn more about Guanine Platelets?

There have been efforts to understand the process of biomineralization of guanine better in order to attempt to mimic it. Current research suggests a regulated pathway involving amorphous precursors and preassembled protein scaffolds in the form of amyloid like fibers, guiding crystal orientation. This biological control enables the formation of uniformly oriented platelets and. In 2023, Hu et al. (2023) have shown that guanine platelets can be reproduced in vitro with comparable size, morphology, and polymorph (mostly β-guanine) to their natural counterparts, using controlled precipitation systems with additives such as polyvinylpyrrolidone or hypoxanthine.

In a synthetic biology context, expressing pathways that increase intracellular guanine availability and promote its crystallization could, in principle, yield cells that accumulate reflective platelets rather than pigments. If successfully implemented, this approach could produce a visually distinct, high-contrast white or iridescent tattoo signal independent of baseline melanin levels. In the future this might successfully address the inclusivity limitations of melanin-based designs.

Hydrogel Encapsulation

The next experimental phase would focus on identifying a biocompatible, transparent, and stable hydrogel that meets our design requirements. Based on expert advice, the most practical start would involve simple gelatin- or alginate-based systems to establish basic compatibility and optical properties. Subsequently, more advanced systems such as Hyaluronic Acid–PEG Click Hydrogels (SPAAC) or Collagen–PEG Thiol–Maleimide Hydrogels Hyaluronic Acid–PEG Click Hydrogels (SPAAC) or Collagen–PEG Thiol–Maleimide Hydrogels would have to be experimentally evaluated for long-term stability and crosslinking performance. Such encapsulation is not only a biological necessity but also a regulatory prerequisite for any clinical translation.

Digital Interface

Although our tattoo allows continuous signal formation, its color intensity cannot be interpreted linearly with concentration by the naked eye. For this reason, a logical next step would be to integrate the system with a smartphone-based readout app, capable of guiding standardized imaging (lighting, distance, exposure) and calculating relative pigment intensity changes over time. This digital interface would enable individualized health tracking while keeping patient autonomy and data sovereignty central. This data can also be easily shared with treating physicians upon request.

Ethical and Regulatory Framework

Because InkSight is designed as a platform technology, capable of adapting to different biomarkers, it carries an inherent dual-use potential. A platform built for flexibility can, by the same logic, be repurposed or misused. Beyond the danger of technical sabotage, there is the subtler risk of functional alteration through imitation and repurposing by third parties, particularly given the modular, swappable nature of our system. Such the system could be adapted to monitor unwanted parameters or exert control rather than provide care.
To mitigate this, we see a clear responsibility to limit access to cell engineering and integration processes prior to implantation. One option, also raised in our ethics meeting, is to establish centralized, certified facilities where InkSight preparations are manufactured and validated. Such a model minimizes unregulated manipulation and ensures traceability. A more detailed assessment can be found in our policy analysis. Additionally, filing a patent would therefore not only serve as a commercial strategy but as a form of governance to prevent uncontrolled reproduction and enforce responsible use.
Beyond misuse in research or production, we also discussed the possibility of coercive or abusive application in a meeting with an expert in medical ethicsexpert in medical ethics. For example, in domestic or patriarchal settings where control over a person’s body or health data could be exerted through such a device. Now, of course this instrumentalization to foster abuse can in theory occur with every medical practice. However, this tattoo has the inherent property of not being easily detachable like many technical deices. Ideally, technologies like ours would always be embedded in therapeutic frameworks that include psychological support and informed counseling. Yet, as we observed in current medical practice, even in sensitive areas such as reproductive medicine, psychological assessment is often not mandatory. Instead, counseling is typically optional. We therefore believe that our system will, realistically, follow a similar model of voluntary but strongly recommended counseling, especially before long-term diagnostic use.

Societal and Economic Implications

When we designed InkSight, our intention was to relieve the healthcare system rather than add to its burden. By enabling targeted, data-driven diagnostics under medical supervision, such a technology could help optimize monitoring intervals and reduce unnecessary examinations. As our exchange with health insurers revealed, reimbursement pathways in Germany are highly centralized and evidence-driven. Beyond regulatory approval, any implementation would depend on demonstrating a measurable clinical benefit for patients and economic value for healthcare systems. Establishing this evidence would therefore be a central goal of any future development phase. If such evidence can be established, the diagnostic tattoo could contribute to relieving the healthcare system by enabling targeted follow-up examinations and personalized monitoring intervals.

In addition to clinical validation, we see a crucial need for public education and societal dialogue. The results of survey form the foundation not only for improving the technology’s practical usefulness but also for gaining insight on the different states of discourse relevant to different groups. Many healthcare professionals we spoke to emphasized that while they see considerable benefits integrating new biotechnologies, they face significant challenges in communicating their principles and safety to patients. In practice, there is rarely time to explain the fundamentals of technologies such as genetic engineering, which often remain emotionally charged and poorly understood.
We therefore believe that a proactive, inclusive dialogue on emerging biotechnologies is essential. Topics such as artificial intelligence have shown that open, critical, and multifaceted public debate, through podcasts, media reports, and accessible expert discussions, can significantly shape acceptance and understanding. A similar culture of discussion must evolve around synthetic biology and medical biointerfaces if such innovations are to be integrated responsibly.

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