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Project Description

To develop an impactful solution, careful consideration of the underlying problem is required. Here, we present why and how we chose our project. We elaborate on the societal issue and how we aim to contribute tackling hidden hunger through ApptaVita.

Challenging hidden hunger

One in three people, an estimated two billion people worldwide, suffer from micronutrient deficiencies, according to the Global Nutrition Report [1, 2]. The high incidence rate of micronutrient deficiencies remains in Sub-Saharan Africa and South Asia [2]. Action has been taken over the years to reduce micronutrient deficiencies across the globe, but progress has been far too slow and unequal across different regions and countries. The United Nations (UN) decided in 2016 that action should be taken towards eradicating malnutrition in all its forms, resulting in a new roadmap towards this goal, the UN Decade of Action on Nutrition 2016-2025 [3].

Micronutrient deficiencies are defined as the lack of vitamins and minerals required by the body in very small amounts. Early symptoms of micronutrient deficiency are often generic and invisible, especially when the deficiency involves multiple micronutrients. These symptoms include reductions in energy levels, mental clarity, and overall performance. Therefore, micronutrient deficiency is often referred to as "hidden hunger". However, long-term insufficient micronutrient intake can have more severe effects on people’s health, such as impairment in growth, increased risk of infectious diseases, and poor reproductive outcomes. Consequently, in the words of the World Health Organization, micronutrient deficiencies contribute to perpetuating a cycle of poverty and ill-health [4, 5, 6, 7].

It is estimated that one in three people suffer from vitamins deficiency and lack access to a proper diet and supplementation.

Image of HPLC

The symptoms mentioned above can be prevented by ingesting the right amounts of micronutrients, but that is not always easy. Vitamin- and mineral-rich foods are often expensive, scarce, or unavailable in low- and middle-income regions. Financial and nutritional resources must be optimized, and well-informed strategies must be made for intervention programs coordinated by health organizations and governments. Therefore, high quality and quantity collection of data on micronutrient deficiency is essential and needs to be improved as reaffirmed in the UN Decade of Action on Nutrition [1, 3, 8, 9]. Over the years, health organizations have set up databases to obtain a quantitative and accurate overview of regional micronutrient deficiencies. Besides this, the databases aim to track progress made in the battle against micronutrient deficiencies and evaluate the current strategies applied by the WHO and other partners. The databases are thus an intrinsically important aspect of eliminating global micronutrient deficiencies [10, 11]. Often the input for these databases is acquired through surveying. Sometimes, data is obtained through analysis of patient samples. Currently, quantitative laboratory tests such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) are used to obtain the required input for the database. These diagnostic techniques are expensive and require skilled personnel, which makes them inaccessible to the majority in low-income countries [12]. This results in a slow-updating database, hence a backlog into monitoring micronutrient deficiencies, resulting in perpetuation of socioeconomic health inequalities due to hidden hunger. This has inspired us to develop a rapid diagnostic test (RDT) that is able to diagnose micronutrient deficiencies in low-income regions . In our project, we focused on Uganda as our target country. To find out more, visit our Human Practices, Integrated Human Practices, and Proposed Implementation pages.

Our solution: AptaVita

AptaVita is a novel and modular aptazyme-based biosensor enabling diagnosis of vitamin deficiencies from blood, thereby taking our first action towards tackling hidden hunger by increasing data availability of vitamin deficiencies. Our biosensor is designed to detect vitamins of interest using aptazymes, ligand-regulated self-cleaving ribozymes. The aptazymes are fused to a reporter gene for a colorimetric read-out on paper support, subsequently quantified by a portable and dedicated hardware. Developing an RDT that is quantitative, accessible, and easily adaptable to other vitamins is a promising approach to fill in the gaps of the missing vitamin deficiency data and build concrete action plans.

Concept workflow of AptaVita. AptaVita's quantiative, accessible, and modular vitamin detection is made possible by 1) use of aptazyme for specific and modular detection; 2) expression of biosensor in a cell-free system on paper support for cheap and accessible RDT; 3) dedicated hardware for robust and quantitative read-out that is portable.

Detection through engineered RNA aptamer-based biosensors

The detection method of specific vitamins relies on aptazymes: synthetic RNA structures consisting of a self-cleaving ribozyme sequence with a ligand-binding aptamer domain [13]. RNA aptamers are capable of binding to specific targets with high affinity and specificity, due to their unique tertiary structure [14]. We have chosen folate (vitamin B9), thiamin (vitamin B1), riboflavin (vitamin B2), pyridoxal (vitamin B6), and cobalamin (vitamin B12) as our target ligands for our aptazymes. To discover these novel aptazymes we used the de novo rapid in vitro evolution of RNA biosensors (DRIVER) [15], a fully automatable selection method that enables multiplexed discovery of biosensors.

The RNA aptazyme-based biosensor consists of two integrated molecular functions: the specific binding of vitamins to the aptamer and the regulation of gene expression by the ribozyme for a colorimetric read-out. In the absence of target vitamins in patients’ samples, the constituent ribozyme allows for self-cleavage of the aptazyme. In its presence, the target vitamin forms a complex with the aptazyme, inhibiting self-cleavage [16]. The aptazyme is fused with a ribosome binding site (RBS) for translation of a reporter gene, lacZ, thus acting as an RNA riboswitch [15]. In a prokaryotic system, constitutive self-cleavage of the aptazyme in the absence of vitamin releases the RBS that becomes accessible for translation of lacZ, resulting in a colorimetric read-out. This system allows for quantification of the amount of the vitamin without the need for laboratory equipment. For more explanation on the regulation of the genetic circuit for colorimetric read-out and on the adapted design for cell-free eukaryotic systems, visit our Design page.

Aptazyme behavior upon absence (left) and presence (right) of target ligand.

High specificity and modularity

Our technology based on aptazymes gives advantages over the use of antibodies to detect small molecules like vitamins and can separate structural analogs [17]. Additionally, the DRIVER method enables the discovery of novel biosensors for a wide range of small molecules [15]. Read more advantages of the DRIVER method on our Design page.

The modularity of our biosensor holds great potential for future applications in the diagnosis of other target ligands by simply re-engineering the aptazyme without changing the read-out system.

Animation of the modularity of the biosensor with re-engineered aptazyme sequence. Other aptazymes developed using the DRIVER method can be re-engineered into our biosensor without any change to the read-out system.

Cell-free expression of the reporter gene

Inspired by the study of Pardee et al. [18] on paper-supported cell-free protein expression, we based the read-out system of our technology on the expression of the lacZ reporter gene in the E.coli-based reconstituted cell-free system, PURE [19, 20]. This cell-free system contains all the necessary components for transcription and translation to express proteins in a GMO-free way. The expression of β-galactosidase from lacZ results in the enzymatic conversion of the yellow-colored substrate, chlorophenol red-β-D-galactopyranoside (CPRG), into the purple-colored product, chlorophenol red. This enables the coupling of vitamin concentration in the sample to the amount of converted product by a colorimetric read-out that is easily detectable. The genetic construct containing the aptazyme upstream of the lacZ reporter gene, CPRG, and the PURE system are freeze-dried on a small piece of paper. This provides support for these components of the biosensor, and ensures easy and sterile transportation [18]. Detailed information about the mechanism behind the β-galactosidase expression in the cell-free system regulated by vitamins can be found on our Design page. Additionally, the results of the experiments regarding the expression of the genetic circuit in cell-free systems can be found on the Results page.

Cell-free expression of biosensor on paper support. All components for expression of the biosensor are lyophylized on paper support. Upon detection of target ligand, expression of β-galactosidase results in the enzymatic conversion of the yellow-substrate (CPRG) into a purple product (CPR).

We used a deterministic kinetic model of our genetic circuit to gain insight into the effect of our system’s components on the discernibility between vitamin concentrations. To evaluate the robustness of this model we performed a sensitivity analysis. Furthermore, we performed a sensitivity analysis on the area between two product curves corresponding to different vitamin concentrations. We used this area as a metric for discernibility. The results from the sensitivity analysis show that the discernibility can be increased, thus providing future directions for improving our system.

Accessible and Cheap

We intended to devise a paper-based detection mechanism that is more accessible and less resource-intensive than current chromatography-based laboratory test methods. The freeze-drying of all system’s components onto paper increases the portability and sterility of the device and allows for easy storage [18]. Furthermore, aptazymes are cheaper than antibodies and can be synthesized in large quantities in a controlled manner [14, 17]. Price comparison with a commercialized rapid diagnostic test was performed in our Entrepreneurship page.

A dedicated read-out device

To accommodate our RDT to be used outside a laboratory environment, we first aimed to design and build a dedicated hardware to quantify vitamin concentrations, which operates independently without the need of other resources such as power supplies. Our hardware measures the color change on the paper discs using the principle of absorbance. This color change is directly coupled to the product formation by β-galactosidase. The absorbance of the product is measured at a wavelength range of 550-600 nm with a peak at 574 nm [21]. A heating system is integrated into the hardware, which keeps the environment at the optimum incubation temperature for gene expression in PURE and for the aptazyme activity [19]. As a result, our hardware is designed for consistent measurement of the vitamin concentration under various conditions. We included a clear manual on how to build and operate the hardware on our Contribution page.

More information on the design and workflow of the hardware can be found at the Design and the Hardware pages.

Dedicated hardware. This allows for a quantitative readout while maintaining an optimal condition of the cell-free expression of biosensor.

Robust, quantitative, and portable

Samples in our hardware are temperature-controlled and shielded from surrounding light. This results in a better resistance against environmental changes allowing for more robust and accurate measurements. The threshold concentrations determining vitamin deficiencies in the blood differ for each person, depending on age, pregnancy, gender, and other health-related characteristics [12]. Hence, we designed our detection system to enable quantitative measurements over a large range of vitamin concentrations for effective interventions to take place. The simple workflow of the hardware requires minimal expertise and training to obtain and interpret results, making it user-friendly. Moreover, compared to standard laboratory equipment, the hardware is small (H: 144 mm W: 140 mm L: 280 mm) and may be used anywhere. More about the decision to quantify vitamin concentrations in blood with a dedicated hardware can be found at the Integrated Human Practices page.

Future prospects

Even with efficient planning during the limited iGEM duration, COVID-19 restrictions still proved to be a significant challenge, leaving us with several future experiments. To reach the goal of accelerating the data collection of vitamin deficiencies, better quantitative measurements are needed. The aptazymes can be further improved to achieve higher sensitivity, selectivity, and binding affinity [15]. We also aim to improve the detection of multiple vitamin deficiencies in a single rehydration step by exploring the liquid flow speed and optimal shape of the microfluidics on a paper-based chip. Furthermore, for blood to be used directly on our RDT, it is required to continue research into the blood plasma separation on paper. The modularity of our technology gives future potential in detecting and diagnosing wide ranges of other small molecules, such as metabolites of prostate cancer. Worldwide, prostate cancer is one of the leading causes of cancer deaths, where recent research has revealed metabolites of many biochemical pathways as cancer biomarkers [22]. Therefore, we believe that our technology will contribute to a healthier future for all and no longer leave anyone behind by limitations of test accessibility.

Other possible applications

• Mycotoxins are one of the most toxic contaminants in food causing a variety of adverse health effects and pose a serious health threat to both humans and livestock [23]. Hundreds of mycotoxins are identified to be present in food and agricultural products. Developing a biosensor that could detect mycotoxins in food could contribute to food security and access to healthy food.
• Hormones control or regulate many biological processes in our body and can therefore be used as a biomarker for some diseases. For instance, higher levels of the estradiol hormone in males can indicate increased risk on male breast cancer development, and developing a biosensor for this could potentially contribute to the early diagnosis of breast cancer in men [24].

References

1. Three issues in critical need of attention. (2018). Global Nutrition Report. https://globalnutritionreport.org/reports/global-nutrition-report-2018/three-issues-critical-need-attention/
2. Hannah Ritchie and Max Roser (2017) - "Micronutrient Deficiency". Published online at OurWorldInData.org. Retrieved from: 'https://ourworldindata.org/micronutrient-deficiency'
3. The Decade of Action on Nutrition 2016–2025 - UNSCN. (2016). Retrieved September 27, 2021, from https://www.unscn.org/en/topics/un-decade-of-action-on-nutrition
4. WHO. (2021, June 9). Fact sheets - Malnutrition. Retrieved September 27, 2021, from https://www.who.int/news-room/fact-sheets/detail/malnutrition
5. Bailey, R. L., West Jr, K. P., & Black, R. E. (2015). The epidemiology of global micronutrient deficiencies. Annals of Nutrition and Metabolism, 66(Suppl. 2), 22-33.
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9. Ambition and Action in Nutrition 2016–2025. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO
10. World Health Organization. NLiS Country Profile. Retrieved September 27, 2021, from https://www.who.int/data/nutrition/nlis/country-profile
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12. Centers for Disease Control and Prevention, World Health Organization, Nutrition International, UNICEF. Micronutrient survey manual. Geneva: World Health Organization; 2020. Licence: CC BY-NCSA 3.0 IGO.
13. Sparkman-Yager, D., Correa-Rojas, R. A., & Carothers, J. M. (2015). Kinetic Folding Design of Aptazyme-Regulated Expression Devices as Riboswitches for Metabolic Engineering. Methods in Enzymology, 321–340. https://doi.org/10.1016/bs.mie.2014.10.038
14. Germer, K., Leonard, M., & Zhang, X. (2013). RNA aptamers and their therapeutic and diagnostic applications. International journal of biochemistry and molecular biology, 4(1), 27–40.
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16. Xiang, J.S., Kaplan, M., Dykstra, P. et al. Massively parallel RNA device engineering in mammalian cells with RNA-Seq. Nat Commun 10, 4327 (2019). https://doi.org/10.1038/s41467-019-12334-y
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18. Pardee, K., Green, A., Ferrante, T., Cameron, D., DaleyKeyser, A., Yin, P., & Collins, J. (2014). Paper-Based Synthetic Gene Networks. Cell, 159(4), 940–954.https://doi.org/10.1016/j.cell.2014.10.004
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21. Kingbright. (2019). WP7113CGCK [Datasheet]. Retrieved from https://eu.mouser.com/ProductDetail/Kingbright/WP7113CGCK?qs=%2Fha2pyFadugjtNsyFL6YAh1DgmyDFXNsj6NJZ3M3NUE=
22. Struck-Lewicka, W., Kordalewska, M., Bujak, R., Yumba Mpanga, A., Markuszewski, M., Jacyna, J., Matuszewski, M., Kaliszan, R., & Markuszewski, M. J. (2015). Urine metabolic fingerprinting using LC-MS and GC-MS reveals metabolite changes in prostate cancer: A pilot study. Journal of pharmaceutical and biomedical analysis, 111, 351–361. https://doi.org/10.1016/j.jpba.2014.12.026
23. Mycotoxins. (2018, May 9). Mycotoxins.https://www.who.int/news-room/fact-sheets/detail/mycotoxins
24. Brinton, L. A., Key, T. J., Kolonel, L. N., Michels, K. B., Sesso, H. D., Ursin, G., Van Den Eeden, S. K., Wood, S. N., Falk, R. T., Parisi, D., Guillemette, C., Caron, P., Turcotte, V., Habel, L. A., Isaacs, C. J., Riboli, E., Weiderpass, E., & Cook, M. B. (2015). Prediagnostic Sex Steroid Hormones in Relation to Male Breast Cancer Risk. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 33(18), 2041–2050. https://doi.org/10.1200/JCO.2014.59.1602

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