Overview

UNIglobin Enzymatic Blood Conversion Kit

In our iGEM project, we have leveraged the glycosidic activity of enzymes derived from Akkermansia muciniphila to cleave the immunogenic A, B, and extended antigens on red blood cells.

As part of this broader initiative, our team developed a hardware solution aimed at detecting residual antigens left over after a round of enzyme cleavage – an incredibly important step for safety before a transfusion, as we found out during our research and in our interviews. While devices for blood typing are already established, they are designed for unambiguous typing, either type A, B, AB, or O [1]. Therefore the development of an approach for detecting the amount of A or B antigen quantitatively is necessary for enzyme conversion. Furthermore, we identified a need to make this device both portable and usable with limited access to electricity, so enzymatic conversion can be possible in the field [2].

To achieve this, we developed the Miniaturized Electronic Antigen Biosensor (MEAB), including a custom printed circuit board to detect antigen levels using voltammetry, with 3d printed housing to use with an accompanying approach utilizing a lectin linked with a redox protein. Additionally to increase the accessibility of enzymatic conversion in the field and the risk of mortality due to leukocyte presence in transfused blood, we developed the UNIglobin Filtration Bag, an inexpensive biological filtration bag that is an alternative to traditional leukoreduction filters using SCOBY, a kombucha precursor [3]. We also included type-specific lyophilized enzyme packs to reduce reliance on the cold chain, the infrastructure required for keeping temperature-sensitive products stable during transportation, which reduces costs of shipping and ensures enzymatic stability long term [4]. To test the sensor, we used porcine blood and our redox-linked lectin to generate voltammetry curves of the reaction to directly evaluate the amount of antigen in a sample of porcine blood before and after enzyme introduction. To test the filter, we established SCOBY’s ability to bind macrophages, T cells, and other leukocytes, and maximized its surface area for leukocyte binding within the bag.

Blood Conversion Kit 1 — **Figure 1:** Rendering of the UNIglobin Enzymatic Blood Conversion Kit

Blood Conversion Kit 2 — **Figure 1:** Rendering of the UNIglobin Enzymatic Blood Conversion Kit

Accessible Enzymatic Conversion of Red Blood Cells

Our project aims to make enzymatic conversion of red blood cells accessible to communities worldwide. We envision the UNIglobin Enzymatic Blood Conversion Kit to be used in blood banks, field hospitals, urgent care centers, offering an affordable and scalable solution for increasing the supply of O blood globally.

In wealthier communities, donors are frequent, and the infrastructure for blood banking and transfusion is well-established [5]. In contrast, lower-resourced communities face limited access to transfusable blood, which is only worsened by incompatible blood types. Lower resource or remote areas may experience power outages, transport delays, or have poor infrastructure, so temperature-sensitive products are less accessible [6]. Our enzymes fall into this category, and thus require a method to keep them shelf-stable for a prolonged period of time. To solve this issue, the UNIglobin Enzymatic Blood Conversion Kit includes shelf-stable packs of freeze-dried enzymes that can last longer than unprocessed enzymes. After enzymatic activity, plasma, leukocytes, residual enzymes, and free sugars are removed via a SCOBY filter: leukocytes bind to the SCOBY, while plasma and enzymes pass through the PTFE membrane and are washed out under positive pressure. ECO RBCs are then collected through the same flow path. Cleavage success is verified downstream using our electronic biosensor, where a lectin-redox complex binds remaining antigens and an electrode measures the redox reaction rate. Peak current correlates with antigen presence, ensuring safety before transfusion; if levels exceed the threshold, the blood is re-treated with enzymes.

Key Values

Design and usage

The UNIglobin Enzymatic Blood Conversion Kit - Your Tool for Converting Blood to Type O

The UNIglobin Enzymatic Blood Conversion Kit provides a safe, effective workflow for enzymatic conversion in the field, equipped with lyophilized enzyme packs, the UNIglobin Electronic Sensor, and SCOBY leukoreduction filters.

What is the MEAB?

The Miniaturized Electronic Antigen Biosensor (MEAB) is a compact, wireless sensor designed for detecting redox reactions (Figure 2). It consists of a 3D printed casing that securely houses a potentiostat printed circuit board, electrode, and necessary reagents. Whether you’re working in medicine, synthetic biology, or chemistry, the MEAB provides you with a platform to quantitatively detect a biomolecule or analyte of choice.

Our sensor is specifically designed for the detection of sugars with the redox-linked lectin our wet lab team developed. Thanks to its simple construction and open-source design, the sensor can be easily replicated and adapted to your specific needs. By lowering the barriers to electrochemical biosensing, we encourage innovation and creative problem-solving.

MEAB 1 — **Figure 2:** Rendering and images of the MEAB

MEAB 2 — **Figure 2:** Rendering and images of the MEAB

How does the MEAB work?

The core of the MEAB is the potentiostat circuit, specifically designed for use with a three-electrode system for precise measurements.

Electronics

The design of the circuit is based off of the NanoStat, a previously developed potentiostat [7]. The circuit (Figure 3) includes an ESP32-PICO-V3 microcontroller with integrated WiFi, 8 bit DAC, and 12 bit ADC for Arduino IDE-based control, the integrated analog potentiostat LMP91000 for cell operation from a single sign power supply, USB-C connection for code uploading, power from a 1.3V lithium-ion battery, and the commercially available MetroOhm screen-printed electrode as the test site. Furthermore, a Wifi antenna is included to be able to run experiments and collect data wirelessly. This printed circuit board is 4cm x 2cm in size, optimized for ease of transport and storage.

MEAB Circuit 1 — **Figure 3:** Image of the MEAB printed circuit board (left). Unaltered Nanostat circuit board within the first casing design iteration (right). From the Nanostat website, the microcontroller and analog front end diagram with each electrode (counter electrode (CE), reference electrode (RE), and working electrode (WE)) (bottom).

MEAB Circuit 2 — **Figure 3:** Image of the MEAB printed circuit board (left). Unaltered Nanostat circuit board within the first casing design iteration (right). From the Nanostat website, the microcontroller and analog front end diagram with each electrode (counter electrode (CE), reference electrode (RE), and working electrode (WE)) (bottom).

Casing

The casing of the MEAB was designed with SolidWorks and developed to protect the circuit, hold the electrode, and carry required reagents. It is made of ASA, a 3D-printed, durable, UV-resistant material that both maximizes how long the components within the sensor can last during transportation and the reproducibility of the device.

MEAB Casing 2 — **Figure 4:** Rendering of the casing for the MEAB (left). Assembled MEAB in casing (right).

Software and Data Analysis

The sensor data is processed in real-time via the ESP32. For more advanced analysis, we use the previously established NanoStat Python software to both connect to and run experiments off of a mobile device. Simply connect the Nanostat to a hotspot or Wifi network to wirelessly run experiments and collect data.

Sensor Usage

To detect blood antigen using the MEAB, a 1 mL sample of ECO RBCs must be pipetted onto the electrode test site. Then the redox-linked lectin in PBS should be placed on the sample of ECO RBCs to let the lectin bind to residual antigens on the surface. Then, after gently washing unbound lectin off with PBS, introduce 1 mL of arginine, a reactant required for the redox reaction, and then run cyclic voltammetry (CV) or altered linear sweep voltammetry (LSV) using the mobile device to generate a curve and detect antigen levels.

What is the UNIglobin filtration bag?

The UNIglobin filtration bag is a leukoreduction filter made of SCOBY, a symbiotic culture of bacteria and yeast [8]. The leukocyte binding capabilities of chitin within yeast give our filter unique leukoreduction capabilities. SCOBY is a purely biological material and is therefore biodegradable, unlike common leukoreduction filters, which are commonly composed of non-biodegradable plastics like polybutylene terephthalate and polypropylene [9]. Since SCOBY relies on surface lectins to bind leukocytes, its efficacy is limited by the surface area that comes into contact with the blood it is treating. Therefore a major design consideration was how to maximize the SCOBY’s contact area. Due to SCOBY’s strong matrix, its shape can be altered depending on its growth surface, so within the bag we placed several folded layers of SCOBY, grown on a folded surface, as well as lining the bag. The output is capped so leukoreduction can be maximized through manual mixing. Furthermore, a small pore-size PTFE membrane is included at the output to catch red blood cells for collection and filter plasma, which may have incompatible antibodies.

Filtration Bag 1 — **Figure 5:** Images of the UNIglobin Filtration Bag

Filtration Bag 2 — **Figure 5:** Images of the UNIglobin Filtration Bag

UNIglobin Enzymatic Blood Conversion Kit Workflow

The UNIglobin Enzymatic Blood Conversion Kit exponentially expands the supply of O blood that can be collected from field transfusions. Type A lyophilized enzyme packs convert type A to type O, type B lyophilized enzyme packs convert type B to type O, and type AB lyophilized enzyme packs convert type AB to type O. These packs also target the extended antigen groups. After rehydration in phosphate buffer solution, the enzyme packs can be connected to the UNIglobin Filtration Bag, where antigens on red blood cells and platelets will be cleaved and leukocytes will stick to the SCOBY. After this the ECO RBCs and platelets can be connected to an external PTFE filter bag for plasma and platelet collection, where ECO RBCs will be caught by the membrane. To ensure the red blood cells don’t aggregate, backflow steps with a buffer into a collection bag should occur every 10 minutes for RBC collection. Once ECO RBCs are collected, a 5 mL sample should be set aside for testing. MEAB works by utilizing a redox reaction detecting electrode circuit with a redox-linked lectin that binds to residual antigens on blood. Refer to Sensor Usage to learn more about the sensor workflow. This process allows us to treat, filter, and validate enzymatic removal of red blood cells. Additionally, the transportability and cost-effectiveness of this system makes it suitable for rapid enzymatic treatment in low-resource settings.

Results

In our project, we successfully detected the reaction of nitric oxide synthase and arginine. Future experiments will test detection of the presence of antigen on porcine red blood cells after treatment with an A-targeting enzyme to verify the sensor’s ability to detect antigen. Further future experiments will vary concentrations of arginine to establish calibration curves indicative of levels of antigen. Through this calibration, we can accurately determine the concentration of antigens. This result confirms the effectiveness of MEAB to precisely detect the rate of redox reactions, which verifies our tool as a platform for redox reaction detection and as a proof-of-concept for antigen detection.

Colorimetric substrates — **Figure 6:** Voltage vs. Current graph using a test solution of CuSO4 and HCl with linear sweep voltammetry on the MEAB. The linear characteristic of this graph verifies that the sensor successfully detects redox reactions.

Figure 7 shows current per unit of voltage vs. time for three conditions - porcine O blood, porcine A blood, and enzymatically treated porcine blood. In a paired t-test, type O (positive control) and treated blood were highly significant compared to type A (negative control) (p<0.001). To simplify analysis, we can calculate a difference in the slopes between the different conditions, with the positive control and treated condition having nearly a two-fold higher slope than the negative control. This was tested at a single voltage sweep between 0 mV and 50 mV with a step size of 5 mV/s - further optimization of this range would give even greater sensitivity, allowing smaller volumes of blood (<10 µL) to quantify antigen presence.

We can see leukocytes bound to the SCOBY surface using immunofluorescence staining with DAPI and anti-CD45 (a marker for most leukocytes) staining on a SCOBY sample (Figure 8A,B). Cells varied in size and morphology, suggesting that bound leukocytes included macrophages and smaller T cells (Figure 8B). However, the number observed was lower than we expected. Further optimization is needed to maximize the effective surface area of SCOBY or to enhance leukocyte binding.

These scanning electron microscopy images show SCOBY treated using different processing techniques- air-drying vs. freezing and lyophilizing (Figure 8). Freezing and lyophilizing SCOBY greatly alters surface topography, increasing the surface area that leukocytes can bind to. Therefore this manufacturing process has the potential to increase the leukoreduction capabilities of our SCOBY filtration bag. Furthermore, we determined that the pore size of SCOBY ranges from 0.2 µm - 1.5 µm, which suggests that SCOBY could be used to filter small proteins and antibodies within plasma, which are typically between 2 to 300 microns in size [10].

Future Work

Future MEAB experiments will vary concentrations of arginine to establish calibration curves indicative of levels of antigen. Through this calibration, we could accurately determine the concentration of antigens. Regarding our filtration system, we will further verify SCOBY’s leukoreduction capabilities through flowing blood through the bag and looking at leukocytes on the SCOBY surface. We will also compare leukoreduction capabilities to traditional leukoreduction filters to compare their efficiency through analysing the treated red blood cell surface. Additionally, we obtained our PTFE membrane from JVLAB but accidentally purchased the hydrophobic version instead of the hydrophilic one required for plasma separation. Due to time constraints, we were unable to replace it, though the design remains compatible with hydrophilic PTFE for future testing.

Outlook

MEAB is easily reproducible, and its application can be easily modified to different applications. Its ability to collect anonymized data could facilitate further research and improve diagnostic accuracy and typing outcomes over time. With the right infrastructure and support, our concept of a kit for enzymatic blood conversion has the potential to significantly increase the supply of O blood. It can bridge the gap between developed countries with established blood supply and infrastructure and blood deserts. As we continue to refine the UNIglobin Enzymatic Conversion Kit, our vision is to transform blood banking and field medicine, making O blood accessible to all, regardless of geographical or economic barriers.

Design Files

https://github.com/rcrane4/UNIGlobin-Hardware

REFERENCES

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