Team:TUDelft/Entrepreneurship

Team

Team Members Collaborations Attributions Sponsors

Project

Project Description Design Engineering Success Results Hardware Contribution Sequencing analysis Partnership Sustainable Development

Lab

Protocols Notebook Parts

Model

Model

Human Practices

Human Practices Integrated Human Practices Education & Public Engagement Proposed Implementation Safety Entrepreneurship

Achievements

Judging Form Medal Criteria Awards and Nominations

Entrepreneurship

This module aims to explore the business potential of AptaVita. Three core aspects of the business were evaluated: the proposed customer, the product, and the company. In these sections, a set of strategic analyses were applied that led to a lean business model based on the business model canvas principle.

Executive Summary

AptaVita is a rapid diagnostic test (RDT) for vitamin deficiency at the point of care. The novel detection technique of the test ensures that it is user-friendly, robust, quick, affordable, modular and deliverable to the end-user. Furthermore, a dedicated read-out device is developed for AptaVita to obtain quantitative results from the test. With these results, healthcare workers can provide personalized dietary advice and make well-informed decisions regarding treatment in severe cases. The generated data can also be shared with existing, open source databases that have been developed by various health organizations. This improves the availability of data on the prevalence of vitamin deficiencies, which can be used as a tool for creating substantiated intervention strategies together with the local government.

By means of a thorough literature research of the local context, the prospective customers of AptaVita were identified as the public and private healthcare facilities in Uganda. Furthermore, we interacted with potential stakeholders of AptaVita to gain more insight in the situation of micronutrient deficiencies in Uganda. Based on this information, a root-cause reasoning established one of the key underlying reasons for the prevalence of vitamin B deficiencies in Uganda. There is a lack of RDTs that are able to diagnose vitamin deficiency at the point of care. A point-of-care RDT must be real-time connected and allow for easy specimen collection. Furthermore it should be environmentally friendly, affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to the end-user [1]. These requirements were adopted by the World Health Organization (WHO) as the REASSURED criteria and present a guideline for the development of new point-of-care RDTs.

The product and its envisioned implementation are designed to minimize the risks established from the risk-benefit analysis. For example, for the collection of the blood sample it is important to assure a minimal risk of contamination and the transfer of blood infectious diseases. Thus, user manuals must be delivered with the product and AptaVita is envisioned to be used in hospitals where trained physicians conduct the test.

Through analyzing the internal operations of AptaVita, three categories of activities were determined that AptaVita will engage in the coming years: the research and development phase, legal admission, and commercialization. With an external business analysis the market of AptaVita was analyzed. It was found that there are few competitors and that these competitors, together with suppliers and customers, have a low to moderate influence on the market. This makes the Ugandan RDT industry for vitamin deficiency an attractive market to enter. Finally, with a business model canvas, the business structure of AptaVita was summarized. It can be concluded that AptaVita technology is especially unique because of its modularity and price, making it an appealing product for our envisioned target group.

Customer

To identify who the customers of AptaVita are, the problem at hand was investigated by diving into the local context and dissecting the problem through a root-cause analysis and by engaging with stakeholders from different sectors (see Integrated Human Practices). It is concluded that the customer segment consists of private and public healthcare facilities.

Local context

It is estimated that one in three people suffers from micronutrient deficiencies worldwide [2]. Micronutrient deficiencies, or hidden hunger, is a form of malnutrition where the patient has lowered levels of essential vitamins or minerals. To further define the scope of AptaVita, we focus on vitamin deficiencies. Symptoms of vitamin deficiencies are often generic and hard to diagnose. Although short-term symptoms are often mild, long-term deficiency can lead to severe health impairments such as blindness, underdevelopment of the brain, and anemia [3, 4, 5]. Over the years, action has been taken to reduce vitamin 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 called the UN Decade of Action on Nutrition 2016-2025 [6].

The highest incidence of severe vitamin deficiencies has been predicted in the sub-Saharan region and South Asia [7]. To narrow the scope of AptaVita, the sub-Saharan region was explored further because of the higher number of recorded disability-adjusted life years (DALYs) due to vitamin deficiencies [7]. To deepen our understanding of the context of the implementation of point-of-care diagnostics in sub-Saharan Africa, we have chosen to focus on one country: Uganda.

To combat vitamin deficiencies, the data on the prevalence of vitamin deficiency needs to be increased. With this data, well-informed strategies can be developed by health organizations and governments to tackle vitamin deficiencies more efficiently. Efforts have been made to increase data availability on hidden hunger, but data is still lacking. Testing of vitamin deficiencies is the first step towards increasing data availability [2, 8, 9].

Current methods to screen for vitamin deficiencies include laboratory tests such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS). These tests can only be performed in laboratories by highly trained technicians and are therefore resource-intensive [10]. This decreases the accessibility of the tests for countries with insufficient economic and logistical means. Fortunately, accessible rapid diagnostic tests (RDTs) provide an opportunity to increase test availability and thus the data on the prevalence of vitamin deficiencies.

Root-cause analysis

The misidentification of the causes of hidden hunger are an integral part of the problem. To identify the origin of the prevalence of vitamin deficiency in Uganda, the ‘5x Why?’ approach has been applied, assisted by the input of various stakeholders and experts. It was found that a lack of data on vitamin deficiency in Uganda is the underlying problem. Thus, AptaVita aims to address this lack of data by developing an RDT for vitamin deficiency that can be performed at the point of care. The root-cause diagram is shown in Fig. 1 [11].

Fig. 1 Tree diagram displaying the root-cause analysis for vitamin deficiencies in Uganda. The method to derive this root-cause analysis was the "5x Why?" method.

No awareness on the importance of micronutrients

By talking with experts on healthcare in Uganda, we realized that awareness of the importance of vitamins and the negative health effects of deficiency is lacking. From media and outreach publications by the Ministry of Health of Uganda, it becomes evident that a lot of time, money, and efforts are put towards combating more visible diseases such as HIV, malaria, and hepatitis B [12]. Besides this, urbanization makes the lack of knowledge on the importance of vitamins more apparent. People that used to live in rural areas are moving to urban areas where the variety of available foods is different from what people are used to. Subconsciously, people in the urban areas are more drawn towards easier accessible food such as rice. This results in less nutrient-rich food patterns [13, 14] [TU Delft, interview Dr. Diehl].

No access to complete and diverse diet

Another general cause of vitamin deficiencies in Uganda is a lack of access to a complete and diverse diet. The use of climate control in agriculture, for instance via greenhouses, enables farmers to produce a variety of crops throughout the year rather than during particular seasons. Although climate control is increasing rapidly and promoted as an essential requirement for farmers that still rely on open-field cultivation, it is not yet a common practice in the Ugandan agricultural sector [15, 16]. Therefore, most farmers are dependent on the local climate for growing their crops, resulting in a limited variety of products available for the Ugandan population [17]. For example, in rural Uganda, the diet mainly consists of plantain, starch roots, and cereals that are grown all year round. These are complemented with seasonal nutrient-rich nuts and vegetables [14]. An example is the Katunkuma vegetable, which is rich in vitamin C, and flourishes in well drained, fertile soil. Therefore, it is hard to grow the crop during the dry season [18, 19, 20]. To complement this seasonal diet, either vitamin and mineral supplements should be consumed, food from other regions should be imported, or local foods should be fortified. However, all these options require sufficient financial and logistical resources.

No access to adequate healthcare

Furthermore, it is clear that one of the root causes for vitamin deficiencies among the Ugandan population is the lack of adequate healthcare. Factors such as high expenses, reduction in stigma due to seeking medical advice (impact on socio-economic status), and lack of resources play a major role. Especially the lack of testing has a great impact on vitamin deficiencies in Uganda. The major issue is that only laboratory tests are available. These tests require skilled personnel, expensive equipment, and proper infrastructure to bring samples to laboratories. To improve on the current techniques, which cannot be applied at the point of care, AptaVita aims to develop a rapid diagnostic test that meets the REASSURED criteria. [1, 21] [MUST, interview Dr. Obungoloch, Mbarara Hospital, interview Dr. Ochora].

Customer needs

Empathy Map

The empathy map is a tool to deconstruct customers’ feelings, experiences, and goals to identify their needs according to the 7 empathy map questions as shown in Fig. 2. Through literature research and by interacting with our stakeholders, we identified two customer segments: the public and private healthcare facilities (see Integrated Human Practices) [22] [GAIN, interview Camp and Cattaneo]. The empathy mapping tool was applied to both customer segments. To access Fig. 2 in higher resolution, please click here.

Fig. 2 The empathy map canvas. The empathy map canvas is a tool to deconstruct customers’ feelings, experiences, and goals to identify their needs according to 7 guiding questions [22].

WHO are we empathizing with?

It is important to identify who exactly the customer is, what the situation is that they are in and what exactly their role is within this situation. In this case, the public and private healthcare facilities are our intended customers, and all have one aim: they want to improve the health of the Ugandan citizens. Therefore, in the scope of vitamin deficiencies, treating and preventing said deficiencies among the population is the main approach to reach this goal. One way to improve the efficiency of both treatment and prevention is early and adequate diagnosis [GAIN, interview Camp and Cattaneo].

What do they need to DO?

Both private and public healthcare facilities will buy our product. Employees of the facilities will then use the tests to screen patients for vitamin deficiencies. Subsequently, the results from the test must be uploaded to micronutrient databases to increase the data availability on vitamin deficiencies. Ultimately, this generated data will assist governments and health organizations to develop large-scale intervention strategies. Furthermore, the data will enable physicians to give personalized dietary advice, and in severe cases immediate treatment.

What do they SEE, SAY, DO and HEAR?

From interviews with Dr. Obungoloch and Dr. Ochora it became clear that there are many Ugandans that are undernourished. Many of these patients show symptoms of micronutrient deficiencies, such as reduction in energy level, mental clarity and overall performance [23]. However, such symptoms are often generic and cannot rule out possible other diseases. Therefore, physicians find it imperative to perform tests for proper diagnosis and corresponding treatment. Currently, very few tests are performed due to a lack of resources such as laboratory equipment and reagents at healthcare facilities.

What do they THINK and FEEL?

Feelings and thoughts of our customer segment can be expressed as pains and gains. The pains that our customer segment experiences, mainly include a lack of resources to provide access to and perform medicine. Often, there are limited laboratory equipment and reagents available at the healthcare centers. This makes coming to the right diagnosis and advising on subsequent treatment challenging, and makes healthcare workers often feel powerless. The gains experienced by our customer segments are directly related to their common goal: improving the health of the Ugandan population. By making diagnostic tools more accessible, AptaVita would add to the gains of its customers [12] [MUST, interview Dr. Obungoloch, Mbarara Hospital, interview Dr. Ochora, GAIN, interview Camp and Cattaneo].

Current tests available on the market

By talking with a biomedical engineer and a doctor at the Mbarara University and the hospital in Mbarara Uganda respectively, Dr. Obungoloch and Dr. Ochora, we found that rapid diagnostic testing for vitamins and minerals is not a common practice in Ugandan healthcare facilities. Instead, current methods applied in Ugandan healthcare facilities are all laboratory tests including methods such as HPLC and MS [10]. Only for vitamin D, the reagents for these laboratory tests are present in hospitals. To test for other vitamins, samples have to be sent out to external laboratories due to a lack of reactants in the local hospital laboratories. Furthermore, these laboratory tests are expensive and require skilled personnel [10]. There have been efforts to produce rapid diagnostic tests (RDTs) for vitamin deficiencies worldwide. Examples are shown in Tab. 1 [24, 25, 26, 27, 28, 29, 30]. From Tab. 1 it becomes evident that there is an abundant offer of RDTs for the detection of vitamin D deficiency. However, for other vitamin targets, only two RDTs were found, one of which is not available on the market yet. All other RDTs currently available on the market are immunoassays, requiring a constant supply of antibodies specifically produced for each test, which is relatively expensive and complicated [31]. Thus, there is a need for an easier and cheaper RDT for the detection of vitamin deficiencies at the point of care.

Tab. 1 Overview of rapid diagnostic tests for vitamin deficiencies. The name of the test is indicated, as well as the test type, for which vitamin it is developed, the price, and its availability.

Name	Test type	Vitamin	Availability	Price per test
NutriPhone (Mehta Research Group) [24, 25, 26]	Fluorescence immunoassay	A, B12, Iron, C-reactive protein	Not available on the market	N.A.
Vitamin B12 (VB12) Rapid Test (Creative Diagnostics) [27]	Immunochromatographic assay	B12	Available on market	Unknown
VHC Quantitative Vitamin D Test (Puremed) [28]	Immunochromatographic assay	D	Available on market	€16,-
AllTest Vitamin D Rapid Test (Suresign Professional) [29]	Immunochromatographic assay	D	Available on market	Unknown
Suresign Finecare Vitamin D Rapid Test (Suresign Professional) [30]	Fluorescence immunoassay	D	Available on market	Unknown

Product

This section describes the product provided by AptaVita. It discusses the key benefits of the test and demonstrates how the test offers value to our customers. Furthermore, product risks are accumulated and classified to be able to anticipate and mitigate potential safety issues. Finally, these are weighed against the benefits of introducing AptaVita in the final risk-benefit balance.

Product description

Private and public healthcare facilities in Uganda are responsible for screening patients for vitamin deficiencies. Current methods of testing are inaccessible, expensive and time-consuming, as they require remote laboratories and trained staff. Our product, AptaVita, offers these customers a quantitative, modular, accessible and affordable diagnostic test to measure vitamin deficiencies at the point of care, allowing them to acquire results within one to three hours.

Fig. 3 provides an overview of the Aptavita workflow. The vitamin levels within a target population are monitored using our diagnostic test. Healthcare workers perform the test by extracting a blood sample from the patient by means of a finger prick and proceeding to place it onto the test cassette. Then, a dedicated read-out device provides a diagnosis and generates the necessary data.

Fig. 3 The envisioned workflow of the AptaVita test. For more information visit the Proposed Implementation page.

The principle of our product is biosensing. By using aptazymes that bind specific vitamins, the test generates a concentration dependent read-out signal. In the absence of vitamins, an aptazyme cleaves itself, while in the presence of vitamins, it forms a complex that inhibits cleavage and stabilizes transcription. This biosensing principle is then used to regulate the expression of a reporter gene, lacZ, using a cell-free system. This way, the use of a whole-cell system can be avoided. For more information, visit the Design page.

The RNA aptamer is a helpful agent to detect vitamins because

• Their unique tertiary structure provides a high affinity and specificity to a specific ligand [31].
• The modularity of the aptamer allows for the detection of new ligands by changing the aptamer without changing the read-out system [32].

The use of a cell-free system has the following advantages:

• A cell-free system contains all necessary components for the transcription and translation to express proteins in a GMO-free way.
• The system is freeze-dried onto paper which allows for easy portability and sterility [33].
• The freeze-dried cell-free system can easily be stored and transported, making it more accessible.
• The use of a cell-free system does not fall under the current GMO regulations [LUMC, interview Prof. Ria Reis].

The safety of AptaVita is guaranteed through the following measures (for a full description see Safety):

• The usage of a cell-free system instead of GMO’s minimizes the biocontamination risk.
• Trained healthcare workers conduct the test in a hospital environment.

Value proposition

The market for our diagnostic test includes private and public health care facilities located in the target country, Uganda. AptaVita creates value for these customers through the following aspects:

• Point of care: The current methods to obtain data on vitamin deficiencies include methods like HPLC and MS. In order to conduct these analytical methods, laboratories and trained clinicians are required. The Healthcare facilities in Uganda performing the test lack the resources to conduct these tests and are required to choose between either outsourcing or not performing the test at all [10]. Our product allows the clinics to complete the test at the facility without outsourcing samples to an external lab.
• Quality: The Healthcare facilities responsible for the implementation consider quality as a minimum requirement as they wish to ensure correct and consistent test results for their patients [1]. Therefore, AptaVita uses a blood sample to detect vitamins to provide accurate and reliable measurements.
• Trustworthiness & Acceptability: For now we have chosen to manufacture our product in the Netherlands due to higher quality and safety control standards. This is further elaborated in our Safety page and in the external business analysis section of this page.
• Affordability: The accessibility of the test for the target groups depends on a low price [34]. Through affordable systems and the multiple vitamins measured on one piece of paper, we have a price that is 70% cheaper than similar tests currently on the market. More information about our prices can be found in the business model canvas.

Risk-benefit analysis

We conducted a risk-benefit analysis and split it into four sections; a sensitivity analysis on the potential risks, a mitigation strategy, and a risk-benefit analysis. Initially, we assess the underlying uncertainties of our test that could negatively impact our business. For each risk, a strategy is proposed to mitigate that specific risk. Finally, we weigh the risk to the benefits of introducing AptaVita in the final risk-benefit balance.

Sensitivity analysis on the potential risks

In this sensitivity analysis, the following risks are identified as potential negative outcomes of the implementation of AptaVita:

• Gain-of-function by ampicillin resistance genes
• Spreading blood infectious diseases
• Misuse of personal data
• Biocontamination of genetic material in the environment
• The barrier to technology diffusion
• Unauthorized reselling

By engaging with stakeholders (see Integrated Human Practices), we classified these risks based on their probability and impact on a scale of 1 to 5, displayed in Fig. 4. The higher the risk scores in these two respects, the more priority the risks should be given in the risk mitigation plan. The explanation of the position of each risk within the matrix is described below.

Fig. 4 Risk matrix concerning the risks of implementing AptaVita. The risks are positioned based on their probability of occurrence and their possible impact.

• Gain-of-function by ampicillin resistance genes:

  The test itself contains plasmids with an ampicillin resistance gene, necessary for amplification during manufacturing of the device. If other microorganisms take up the plasmid, it can cause resistance to the medicine.

  • Probability (1): The plasmids are present in very low concentrations, so the transferability rate is trivial. In addition, the plasmids contain a pMB1 E.coli origin, which makes the replication in other microorganisms besides E.coli unlikely, even if it is taken up.
  • Impact (3): If a microorganism takes up the plasmid, it could cause resistance to the medicine. The resistance would impact the individuals infected by an ampicillin-resistant microorganism, but there are many alternatives to treat infections like these.

• Spreading blood infectious diseases:

  The test requires blood withdrawal through a finger puncture. The use of blood always carries the risk of spreading infectious diseases.

  • Probability (3): Although working with blood has become safer over the years when obtaining samples, the risk of transmission is still present [35]. Especially in Uganda, where biosafety and biosecurity are at 33% for both the private and public healthcare sector and where village health care workers are generally not trained in working with blood [36].
  • Impact (5): The list of infectious diseases that can be spread in suboptimal conditions through blood includes HIV, hepatitis C, and malaria [37]. These are viral infections that have a detrimental impact on a patient's life and can even cause death.

• Misuse of personal data:

  The vitamin deficiency database has a dual-use character. Although it provides crucial information for health organizations, it could also be used for different means.

  • Probability (4): Low- and middle-income countries experience a rapid integration of digital tools in the health care sector [38]. As digital health databases are particularly vulnerable to data leakage due to the sensitivity of the data the probability of occurrence is high.
  • Impact (4): The misuse of health data could give rise to discrimination determining someone's eligibility for employment, housing or other services. A micronutrient deficiency can indicate the chances of someone getting ill and, therefore, unfavourable for a future employer [39].

• Biocontamination of genetic material in the environment:

  The plasmid used in our system could be released into the environment and have unintended effects. This comes with concerns as there are currently no ways to retrieve such engineered entities from the environment.

  • Probability (1): As mentioned for the gain-of-function by ampicillin resistance genes, the transferability rate of the plasmid is trivial.
  • Impact (4): It is hard to assess what effect it might have when the genetic material of our system is integrated into the environment due to the inherent complexity of nature.

• The barrier to technology diffusion:

  End-users may be discouraged to implement AptaVita as they lack the knowledge and awareness of the technology.

  • Probability (2): If AptaVita works as intended, its advantages outweigh the current methods which reduce the chances of end-user rejection.
  • Impact (2): The primary effect is felt by our own business. However, in the long term, vitamin deficiency will remain undiagnosed for the local population.

• Unauthorized reselling

  Our test is solely sold to the healthcare facilities that are our main customers. Distribution of the test to other individuals/parties that are initially not well informed or advised about the test can lead to increased safety risks.

  • Probability (2): As our product is only sold to certified healthcare facilities and requires a dedicated read-out device, we expect the level of unauthorized reselling to be low.
  • Impact (3): If the test is sold to individuals that are initially not well informed or advised about the test can lead to safety issues. These issues include the increased spreading of infectious blood diseases or incorrect disposal of the AptaVita test. This increases the risk of biocontamination and infectious disease spreading.

Mitigation plan

A risk mitigation plan is required to prepare and lower the risks posed by the introduction of AptaVita.

• Gain-of-function by ampicillin resistance genes and biocontamination:

  To avoid that genetic material is released into the environment we must ensure that AptaVita is correctly disposed of. To do so we provide bins and a disposal manual to make sure that all biological material is collected and treated in the same way.

• Spreading blood infectious diseases:

  Manuals should be distributed and trained personnel should perform the blood withdrawal with sterile needles. To further mitigate this risk and to ensure the safety of both patients and personnel, we have decided on the implementation of AptaVita within healthcare facilities. The full argumentation can be read in the Proposed Implementation section.

• Misuse of personal data:

  To prevent the misuse of personal data, this data is stored and shared anonymously. The only data that is stored is the age, gender and vitamin status of a patient. The patient receives treatment directly from the healthcare worker and the data is only relevant to determine high-risk areas that require more attention to prevent vitamin deficiencies.

• The barrier to technology diffusion:

  To increase knowledge and awareness on our test, details on the use and the possible benefits should be transparent through open science practices and a straightforward website. Moreover, the education of the local population on the benefits of conducting the test is of great importance.

• Unauthorized reselling:

  To avoid that our test is sold to unauthorized individuals, there must be a clear protocol to monitor the sales of AptaVita. The test requires dedicated hardware to generate accurate and reliable test results, which makes this monitoring easier because the use of either part is mutually dependent on the other.

Risk-benefit balance

Benefits of applying AptaVita compared to current methods:

• Rapid:

  A drawback of current methods is the time required to perform the test. Healthcare workers can complete our test in 1 to 3 hours, providing direct feedback to patients.

• Affordable:

  By making our test 70% cheaper than our leading competitors, we can increase accessibility and lower the threshold to receive a diagnosis.

• Accessible:

  Our test is conducted at the point of care. This eliminates the need for an external laboratory to which the sample has to be sent.

The AptaVita test is an accessible, affordable, and rapid diagnostic test for vitamin deficiencies at the point of care. There are, however, some risks for which the impact outways the benefits. The spread of infectious diseases, for example, would be an unacceptable result of our test. We should note that if the steps proposed in the mitigation plan are strictly followed, the probability of these risks would be minimized so that the benefits outweigh the risks. Although important to consider, the other risks are less impactful when compared to the described benefits.

Company

By analyzing the internal operations and conducting an external business analysis of the market of AptaVita, all strengths, weaknesses, opportunities and threats were identified and summarized in a SWOT analysis. From this review of the internal and external factors that influence AptaVita, a lean business model was created based on the business model canvas [75]. It can be concluded that AptaVita technology is especially unique because of its modularity and price, making it an appealing product for our envisioned target group.

Internal operation

Patenting

From the patenting guide of the 2020 Leiden iGEM team and during our conversations with V.O. Patents & Trademarks, we found that when a product or idea is novel and innovative (and this can be proven), the creator can apply for a patent. In the case of AptaVita, one of the characteristics of the biosensor is a property that is enriched through the patented DRIVER procedure [40]. This particular characteristic is therefore adopted from a patented method, which poses the risk that a license must be acquired from the owners of this patent upon commercialization of AptaVita. Nonetheless, we were advised to file for a priority patent application if there is a desire of commercializing AptaVita. This priority patent application should specifically describe the aptamers found from the DRIVER procedures, and the aptazyme design that results from this aptamer. With this application, the AptaVita team is able to conduct additional research and development of these specified aptamers and aptazymes for a period of one year. After this period, the application can be ‘polished’ and these results could be included in the official patent.

Manufacturing

For the manufacturing of AptaVita, eventually we aim to produce the key biological components (the cell-free system and plasmids) through an in-house biochemical process using genetically modified organisms (GMOs). The in-house production of a PURE system will require a higher initial capital investment in equipment. However, this approach benefits the overall profitability of the project by significantly decreasing the highest cost drive of our product. As demonstrated by the EPFL 2019 iGEM team, the cost of a PURE system can be reduced up to 0.55 EUR per reaction. To read more about the cost structure, please review the business model canvas below.

Both Europe and Uganda were considered as locations for the production of AptaVita. Even though local production improves the social and economic sustainability of the Ugandan communities, Europe was chosen as the location of production of the RDTs due to safety considerations. In Uganda, currently, a regulatory framework for the use of GMOs is still pending [41]. As mentioned above, GMOs are required to reduce the costs of AptaVita in the long-term. Thus, to ensure the safety of the production process, our manufacturing employees, and the test itself, remote production of AptaVita is preferred. However, as shown in our political, economic, social, technological, legal, and environmental (PESTLE) analysis, we propose a relocation as soon as circumstances allow for it.

Distribution

For the distribution of AptaVita, we would ideally make use of the existing transportation infrastructure. The Ugandan Ministry of Health announced in April 2021 that at least 86% of the Ugandan population is within a 5 km reach of either a public or private healthcare facility [42]. Furthermore, healthcare facilities are supplied with standard equipment such as disposable gloves, needles, and bandaids [43]. Also, RDTs for other diseases such as malaria and Covid-19 are being conducted throughout the population at a large scale [44, 45].Therefore, we hope to be able to use the infrastructure that is already in place for these activities to distribute the AptaVita RDTs. Furthermore, by freeze-drying the paper discs no cold transport is required. This means that no expensive equipment is required for the distribution of AptaVita, which reduces the costs of transport significantly and makes AptaVita more accessible [46].

Compliance

To launch AptaVita on the Ugandan RDT market, our test and read-out device must comply with local laws and regulations. Furthermore, we aim to make AptaVita a modular test that is not limited by the geographical boundaries of Uganda. Therefore, AptaVita should abide to regulations that are upheld by the European Union (EU) and the United States of America (USA). To get approval from all the regulatory bodies, certifications must be acquired that guarantee the safety of the different components of our product. An overview of the most pressing regulations and certifications can be found in Tab. 2 and Tab. 3.

Tab. 2 Overview of main certifications and regulations in Uganda that apply to AptaVita.

Certification	Regulatory body	Type
Ministerial decree ADM.140/323/01 of 20th July 2020 [47]	National Drug Authority (NDA)	Medical devices
Statutory Instrument no 77 of the Surgical Instruments and appliances Regulation 2019 [47]	National Drug Authority (NDA)	Medical devices
Data Protection and Privacy Regulations, 2021 [48]	National Information Technology Authority Uganda (NITA-U)	Data protection and privacy

Tab. 3 Overview of main certifications and regulations in the EU and USA that apply to AptaVita.

Certification	Regulatory body	Type
FDA approval [49]	U.S. Food & Drug Administration FDA	Food, drugs, and medical devices
CE Marking [50, 51]	European Economic Area (EEA)	Safety, health, and environmental protection
FCC certification [50, 52]	Supplier's Declaration of Conformity (U.S.)	Electromagnetic radiation
IEC 62133 [50, 53]	International Electrotechnical Commission (IEC)	Lithium-free batteries
RoHS [50, 54]	European Parliament (EU)	Lead-free products

Marketing strategies

This section describes how we intend to promote the implementation of the RDT into the existing market for the point-of-care rapid diagnostic tests in Uganda for the detection of vitamin deficiencies.

The first step is to gain the approval of our test by the Ugandan government. This is not only required by law but also raises our trustworthiness once introduced to the market. For approval, we must adhere to the requirements and regulations of the Ugandan National Drug Agency (NDA) and the National Information Technology Authority Uganda (NITA-U), see the compliance section.

When we are granted all necessary certifications, we can enter the market and target our customers; public and private hospitals. To effectively do so, we need to partner up with health organizations currently active in Uganda. These organisations allow us to harness their existing connections and enter the market effectively.

Furthermore, based on the village healthcare worker survey that showed us that village communities are generally unaware of vitamin deficiencies. We have decided that our initial target group should be women and children because local doctors informed us that the population is in general most concerned with the health of this group.

Finally, to sell our product and reach our target customers, we will employ a technique known as free sample marketing [55]. Our product consists of a one-time use test cassette and a dedicated read-out device. By providing our prospective customers with one free read-out device, we get the product into the hands of the end-users. This could subsequently boost the complementary test cassette sales as they are required to conduct the test [56]. Although the test cassettes are sold at a lower price than the read-out device, the free read-out device is paid back after a certain number of test cassettes are sold. Additionally, users would have to buy an extra read-out device to scale up their operations.

Project plan

The expected time required for the rollout of AptaVita is displayed in the Gantt chart in Fig. 5. For this, we considered three categories: (i) the research and development phase, (ii) the legal admission, and (iii) the commercialization of AptaVita. To access Fig. 5 in higher resolution, please click here.

Fig. 5 Gantt chart. The Gantt chart shows the planning of the development of AptaVita. Three categories are shown, being the research and development phase, the legal admission, and the commercialization.

Research and development phase

Optimization of the aptamer is required. This entails engineering of the aptamer to realize a sufficient specificity for the vitamin in question and selectivity in complex samples. Optimization is realized by continuing the in vitro evolution experiments. By investing in robotics, the optimization can be realized in a short time [57]. Furthermore, optimization of the ribozyme is required to ensure that the test is rapid. This is conducted parallel to the optimization of the aptamer. The optimized aptamer should be tested with the optimized ribozyme.

The reaction conditions also require optimization. The DNA concentration affects the duration of the reaction and other species such as ion concentrations may affect the output of the assay. This should be conducted with the optimized aptazyme, and thus conducted after the previously mentioned optimization and verification steps. A further elaboration on this parameter optimization can be found on the Modeling page and the Results page.

Besides the ribozyme and the reaction conditions, optimization of the dedicated hardware should also be conducted. The hardware should be designed in such a way that it allows for testing with multiple test cassettes, integration of a power supply, and printed circuit boards, resulting in an overall smaller device. The smaller the device, the more efficient logistical operations. In addition to this, the hardware should be developed further so that it contains a cooling device to allow for testing in areas with a higher ambient temperature than 37 °C. A detailed description of the hardware is found on the Hardware page. Finally, microfluidics should be integrated into the test cassette and hardware, allowing for testing of multiple vitamins with a single sample application step.

Legal admission

The company registration can be conducted in the first quarter of 2022. By talking with a patent lawyer, we found that application of a patent with priority must be done before sharing key technological details. This priority patent will last for one year, in which further research can be conducted to optimize the technology that is claimed in the patent. To obtain legislative approval from international and national bodies for the use of AptaVita as a rapid diagnostic test, the test should undergo clinical trials [58].

Commercialization

Funding is required to commercialize our product. The time required for funding is dependent on the interest of investors and this process can already be started from the beginning. Besides funding, agreements with producers of the test components and distributors are required. Agreement talks with producers and distributors can start just before finishing the research and development phase. The actual production, packaging, and rollout starts after these agreement talks. In the first quarter of 2025, we aim for an official rollout of the product. For this initial rollout, we intend to acquire the Mbarara Regional Referral Hospital as our first customer because of our previously established relationship with them. This year we intend to sell 100 tests to our first customer.

External business analysis

Market analysis

Porter’s five forces analysis is a tool to describe the competition of a market. There is direct competition with rivals within the industry, where competitors jockey for a market position and profits. Furthermore, there are four other forces that influence this rivalry: the threat of new entrants into the industry, the threat of substitute products or services, the bargaining power of consumers, and the bargaining power of suppliers. Fig. 6 shows the influence each of these forces has on the existing industry AptaVita aims to enter [59, 60].

Fig. 6 A graphical representation of Porters’ Five Forces analysis on the prospective market of AptaVita.

Industry rivalry

The industry rivalry within the market that AptaVita aims to enter - the point-of-care rapid diagnostic test industry in Uganda for the detection of vitamin deficiencies - is relatively small. From interaction with stakeholders in Uganda, we know that testing at the bedside of the patient for vitamin deficiency is not a common practice [Mbarara Hospital, interview Dr. Ochora]. Vitamin D can be tested for at the laboratory of the hospital. However, for all other vitamin tests, samples have to be sent to remote laboratories. Here, high-performance liquid chromatography (HPLC) and mass spectrometry (MS) are used to identify the blood vitamin level of patient samples [10]. Thus, competitors within the vitamin deficiency diagnostics industry in Uganda mainly consist of suppliers of laboratory equipment used for testing. From literature research, we know that there are also rapid diagnostic test (RDT) producers that offer RDTs for vitamin deficiencies. These competitors mainly produce vitamin D tests, only one RDT for vitamin B12 was found that is available on the market. All RDTs were immunology-based assays [24, 25, 26, 27, 28, 29, 30]. For more information about competitive tests, please view the Customer section of this page. AptaVita distinguishes itself from these two types of competitors because it is more accessible, affordable, modular, and quantitative. This ensures that upon entry into this market, the rivalry between AptaVita and its competitors may be sparked by this difference in products [60].

Threat of new entrants

The threat of new entrants into our prospective market is extremely dependent on the barriers that prevent entry into this industry [60]. Barriers that prevent competitors from entering the vitamin RDT market in Uganda are listed below. Due to these entry barriers, the threat of new entrants into the industry is low.

• Capital requirements: The research and development (R&D) that is required to create novel rapid diagnostic tests to detect vitamin deficiencies at the point of care is expensive. The capital required to fulfill R&D needs, although different for every technological approach, is high for medical innovations compared to other technological innovations. Furthermore, getting the right certifications and approval from regulatory bodies is a tedious and resource-intensive process.
• Cost disadvantages: The potential new entrants into the industry require specific proprietary diagnostic technology to compete with the existing market. To obtain this, they must go through the previously mentioned approval and patenting process.
• Access to distribution channels: The infrastructure that is present for supply, use, and disposal of RDTs is partly present. We know from public outreach of the Ministry of Health that "approximately 86% of the Ugandan population is within a 5 km reach of either a public or private healthcare facility" [10]. However, available infrastructure for the supply and disposal of the test must be further investigated.
• Government policy: Medical innovations have to be approved by governmental and/or international institutions. To get this approval, certifications that agree on the safety of the product must be acquired. The specific certifications required for medical technologies in Uganda are discussed in Proposed Implementation.

Bargaining power of consumers

The group identified as AptaVita’s customers are private and public healthcare facilities. However, we envision the test to be cheap enough so our customers can charge patients for the test. Thus, we aim for an affordable test that is accessible to the general public regardless of socio-economic status, reducing dependency on charity or health insurance. Therefore, we must keep the price of our product low. Furthermore, we aim for customers to purchase large batches of the tests per order per facility. These two criteria increase the buyers’ bargaining power. However, because AptaVita has a unique test that is not available on the market yet, the bargaining power of customers is decreased. This makes the influence of the bargaining power of consumers on the industry moderate.

Threat of substitute products or services

A potential substitute product could be an actual self-test for vitamins. Currently, we need the test to be conducted at a healthcare facility, where a trained healthcare worker can obtain a blood sample and analyze this with a dedicated readout device. A self-test would make testing even more accessible because these can be conducted anywhere and by the patient themselves. However, for a company to develop and implement a self-test for vitamin deficiency that meets the sensitivity, specificity, accuracy, and quality requirements, the above-mentioned entry barriers must be overcome. It should be noted that these hurdles are easier to overcome by companies that are already into other types of RDTs (e.g. Abbott, BD, QuantumDx) [61, 62, 63]. These companies have experience with the development and distribution of RDTs. Furthermore, financial barriers are lowered because they have already established a market position for other RDTs. This makes the threat of substitute products moderate.

Another potential substitute of RDTs for the detection of vitamin deficiencies is increased access to an adequate diet. This would make testing for vitamin deficiency obsolete. However, we do not view this as a risk, since AptaVita is a means to reach this goal.

Bargaining power of suppliers

There are 4 major resource groups that AptaVita is dependent on which needs to be provided by suppliers:

• Substrate for GMOs that produce cell-free system and plasmids
• Filter paper for paper-based system
• Electronics for the hardware
• CPRG (substrate for enzymatic color change)

For substrate, filter paper, and electronics, AptaVita will not be one of the biggest customers because there are many industries that purchase these goods. For example, the production of many consumables such as beer and vitamins relies on the use of microorganisms, and therefore substrates for these microbes [64, 65]. This increases the bargaining power of the suppliers. However, since there are so many industries and customers that rely on these products, there is more than one company that supplies these goods. This makes the bargaining power of these suppliers moderate.

CPRG is a more specific product that needs to be acquired through external suppliers, making the bargaining power of this specific supplier higher than other suppliers. To reduce this risk, in the future AptaVita could look into the advantages and disadvantages of in-house production of CPRG.

PESTLE analysis

For AptaVita design and proposed implementation choices were made based on interaction with stakeholders. Political, economic, social, technological, legal, and environmental factors can affect these particular choices. To evaluate which choices have a high probability of being influenced by these factors, a PESTLE analysis was conducted [66]. It was found that the decision of using a single-use product, using a cell-free expression system, and producing the tests in Europe are at high chance to be affected by these external forces.

The external landscape of AptaVita

The political, economic, social, technological, legal and environmental influences on the rapid diagnostic testing market are summarized in Fig. 7.

Fig. 7 PESTLE analysis. Political [41], economic [67, 24, 25, 26, 27, 28, 29, 30], social [68] [Mbarara Hospital, interview Dr. Ochora, LUMC, interview Dr. Bengtson], technological [24-30], legal [41] (see Implementation page), and environmental [69] (PESTLE) factors that influence the market that AptaVita aims to enter.

Single-use products

The United Nations (UN) are promoting responsible consumption and production through Sustainable Development Goal (SDG) 12. Avoiding single-use products and disposables is one of the strategies to achieve this goal [69]. AptaVita relies on the use of single-use disposable parts to prevent any contamination with patient samples or the spread of biological parts. This is in conflict with SDG 12 and therefore contradicts the recent environmental, political, and legislative advances regarding sustainability. However, when evaluating the social impact of rapid diagnostic tests (RDTs) over these issues, it was found that governments are currently prioritizing the social impact. RDTs for a variety of other diseases, which are also made of single-use parts, are being distributed to improve the health of the population [44, 45, 70]. Ultimately, diagnosing vitamin deficiency will enable physicians and patients to take action to improve the patient’s well-being, and thereby resolve symptoms that originate from vitamin deficiency. These symptoms include reductions in energy level, mental clarity, and overall performance [5]. These conditions lead to a decreased socioeconomic status. According to the WHO, improving these conditions, therefore, breaks a perpetuating cycle of poverty and ill-health across the world [68].

Cell-free expression system

The use of a cell-free system was chosen over the use of genetically modified organisms (GMOs) for expression because of safety considerations. In the Netherlands, the legal frameworks have strict regulations regarding the use of GMOs outside of controlled research environments. However, the use of synthetic genetic constructs (plasmids) in combination with cell-free systems doesn't fall under the current regulations [RIVM, interview Dr. Van der Vlugt-Bergmans, Dr. Agterberg, and Krouwel]. We expect a similar scenario to occur in Uganda, where a bill for the regulation of GMOs is currently under revision and approval process [41]. Thus, AptaVita is not over-restricted in the development of its technology, which provides a favorable scenario to enter the market. We are aware of the important role that regulation plays to ensure the safe and responsible implementation of technology. We will actively seek to follow alternative legal frameworks that are applicable to our diagnostic test. Therefore, to ensure that the safety of our test is guaranteed, we aim to obtain the certifications and approval required for medical devices (see the Proposed Implementation page). In addition, as relevant actors in this field, we look forward to entering a conversation regarding the development of future regulations on cell-free systems with local authorities.

Production in Europe

For the implementation of AptaVita, we have decided to produce the test in Europe because of the regulations on the use of GMOs, intended for the production of cell-free systems and plasmids [41]. However, through AptaVita we do not only want to bring change to the healthcare sector. Eventually, we also want to promote social and economic development in Uganda through local production. To reach this goal, it is essential to create a network of involved stakeholders from the beginning. This will enable us to move production from Europe to Uganda as soon as safety regulations allow for it. Therefore, upon implementation of AptaVita, we will try to engage with local diagnostics companies such as Astel Diagnostics [67].

SWOT analysis

After establishing all internal operations and conducting an external business analysis, the strengths, weaknesses, opportunities, and threats of AptaVita can be summarized in the so-called SWOT matrix. The strengths and weaknesses describe the pros and cons of the internal operations of the business, whereas opportunities and threats describe the external business environment and its advantages and disadvantages. This SWOT analysis is presented in Fig. 8 [71].

Fig. 8 SWOT analysis.

Business model canvas

The TU Delft 2021 iGEM team aims to develop a rapid diagnostic test (RDT) and dedicated read-out device to diagnose micronutrient deficiency at the point of care. By developing and implementing the test, we aim to improve data availability of micronutrient deficiencies across the world. With this data, the efficacy of current intervention programs can be improved, resulting in better global health [2]. A business model is created to analyze the potential of the start-up "AptaVita". This section describes our dynamic business strategy by using the business model canvas template. The business model of AptaVita applies to Uganda: a country where micronutrient deficiency is highly prevalent [72, 73].

The business model canvas is made up of 9 different sections. At the heart of the business model stand the key propositions of our product. On the right of the canvas, the sales are described by the customer segments, customer relationships, channels, and revenue streams. On the left, the internal operations are represented by the key partners, key activities, key resources, and the cost structure [74, 75, 76].

With this business model canvas, we compare the different possible scenarios for the implementation of AptaVita and conclude that AptaVita will produce its product in high-income countries and sell its product to middle- and low-income countries. To access Fig. 9 in higher resolution, please click here.

Fig. 9 Business Model Canvas of AptaVita.

Key propositions

AptaVita is a rapid diagnostic test (RDT) to detect vitamin deficiency at the point of care, which is robust, user-friendly, affordable, and deliverable to the end-user. These are important criteria that were adopted by the WHO from Land et al. [1] that serve as a guideline for RDTs. The biosensor is designed to be modular and is therefore easily adaptable to other biomarkers. By using a dedicated read-out device we aim to deliver quantitative results. To improve on the availability of prevalence data of vitamin deficiencies, data can be anonymized and shared with existing databases. This way health organizations and governmental institutions are enabled to set up better targeted and well informed intervention programs. Furthermore, the information can be immediately used by healthcare workers at the point of care to provide patients with personalized dietary advice and in very acute cases treatment. Thus, the use of AptaVita will add to the overall health and well being of the Ugandan population.

Profit centers

Customer segments

A literature study was performed, and various interviews were conducted with health organizations and other experts on healthcare and diagnostics in Uganda. It was found that healthcare facilities are divided into public and private institutions, where the government (financially) supports the former. Therefore, these two types of healthcare facilities are our envisioned customer segments [MUST, Interview Dr. Obungoloch, Mbarara University, Interview Dr. Ochora, GAIN, Interview Camp and Cattaneo].

Channels

The channels to reach out to the health organizations include cold calling, the Innovative Food Systems Solutions (IFSS) portal established by the Global Alliance for Improved Nutrition (GAIN) [77], and having an active website and social media. Furthermore, health organizations work closely together with the local government, to assist with setting-up intervention strategies and programs. Also, they advice governments on the availability and quality of medical devices such as RDTs. Thus, establishing a relationship with one or more organizations active in Uganda will aid us in contacting the Ugandan government [GAIN, Interview Camp and Cattaneo].

Customer relationships

We envision three key moments of interaction between AptaVita and its customers: during sales, before use and during use. To assist our customers each step of the way, we aim to set up a sales helpdesk, training upon request and a technical support desk. These services must be located in Uganda to ensure a quick response, and avoid communication barriers.

Cost centers

Key partners

The first two key partners of AptaVita are health organizations and governments. They are especially important as they are the key to approving and promoting AptaVita among our intended customer segment. The relationship between AptaVita and these two partners is further elaborated in the Proposed Implementation page, and marketing strategies including these two partners can be found in the Internal Operations section. Other key partners of AptaVita are distribution companies, investors and suppliers. The distribution of AptaVita does not require complicated procedures for transport since the test is freeze-dried, so AptaVita can make use of the general facilities of transportation companies. Investors are needed to cover the additional research and development phase and the initial start-up cost, which is further explained in the following section regarding cost structure and revenue streams. Finally, AptaVita interacts with suppliers to acquire the right resources to produce the tests. Thus, suppliers can have a significant influence on AptaVita, which is further elaborated in the market analysis section in the External Business Analysis.

Key activities

The key activities AptaVita engages in are directly related to the key partners ofAptaVita. These activities include production of the tests, research & development, sales, marketing, fundraising, and interaction with its key partners. The project plan in the Internal Operations section further elaborates on the timing of these and other activities over the coming years.

Key resources

Key resources for the production of the test cassette are the filter paper, the cell-free system, the genetic constructs (plasmids), and the substrate (CPRG). Key resources that are required for the hardware include electronics, a chip, a power bank or battery, and the plastic casing. However, as explained in the Proposed Implementation page, to perform the test additional equipment such as a lancet or safety needle and Paperfuge or similar device are required to collect the patient sample [78, 79, 80]. Therefore, these must be supplied with the test. For the initial phase of AptaVita, the production of these additional items is outsourced. Other resources that are required to protect intellectual property and realize AptaVita are patents, funding, a website, and a webshop.

Upon scaling up, we aim to produce the cell-free system and plasmids in-house. This requires heavier equipment for the bioprocess and downstream processing. To run these facilities additional resources such as a substrate for the organisms, employees, and electricity are needed.

Cost structure and revenue streams

The costs of AptaVita arise from the manufacturing of the test, continuous research and development, transport, and other miscellaneous activities. To break even and eventually earn profits, these costs must be covered by the revenue streams that are created by AptaVita. The manufacturing costs can be divided into two categories: costs of the test cassette and costs of the hardware. An estimation of the costs of the test cassette was made by considering the four main components of the test: the cell-free system, the filter paper, the plasmids, and the CPRG. A summary of each of these components’ prices can be found in Tab. 4. The first version of the test is produced with the help of a commercially available cell-free system, and commercially acquired plasmids. The cost per reaction for this version accumulates to 4.89 EUR per reaction. The next step to reduce the cost of the test is to produce the cell-free system in-house. For this, the OnePot PURE system as applied by the EPFL 2019 iGEM team is ideal, as the costs for the production of this cell-free system are estimated at 0.09 USD per µL. This results in a total cost of 1.10 EUR per reaction, which is a cost reduction of approximately 78%. Finally, the plasmids could be purified from GMOs instead of ordered commercially. By doing so the final cost of the product becomes approximately 0.77 EUR per reaction. The costs of the prototype of the hardware can be found in Tab. 5. An estimation of the costs of the additional equipment required for sample collection can be found in Tab. 6.

Tab. 4 Cost of components test cassette per reaction.

Component	Costs (EUR)
PURExpress (NEB) commercial cell-free system [81]	0.47
OnePot PURE (EPFL 2019 iGEM team) non-commercial cell-free system	4.48
Whatman grade 42 filter paper [82]	0.01
Plasmid (Twist Bioscience)	1.86
Plasmid purification (Promega PureYield™ Plasmid Miniprep System) [83]	0.14
Total based on commercial kits only	4.89
Total based on OnePot PURE & commercial DNA	1.02
Total based on OnePot PURE & purified DNA	0.69

Tab. 5 Cost of the components of the hardware prototype.

Component	Costs (EUR)
Rasberry Pi	73.23
Other electronics	232.41
Powerbank	45.99
3D printing	100
Total	451.63

Tab. 6 Cost of the additional equipment required per test.

Component	Costs (EUR)
Needles, pipettes, etc. [78, 28]	0.42
Paper centrifuge [79, 80]	0.2
Total	0.62

Other costs that are directly related to the product are due to transport and packaging. These costs must be covered with the revenue streams of AptaVita, which are created through sales of the product. Usually, it is advised for start-ups to calculate about 50% to 60% margins on the total costs of the product [84]. Since the exact costs related to these activities are hard to establish in the early R&D stage (current stage of AptaVita), this margin was increased to 100% of the above-calculated cost of the test cassette and additional equipment. For the hardware, after comparison, 50% margins were chosen because the costs mentioned here are for the proof-of-concept prototype since we expect a significant decrease in the costs for the production of the final hardware. Furthermore, the prices of the AptaVita test cassette for both single reaction and multiplex testing (four reactions) and hardware were compared to a product of a competitor. These results can be found in Fig. 10 and Fig. 11. From this, it can be concluded that the AptaVita diagnostic technology is only 30% of the price of technologies that are currently available on the market, and therefore more affordable. The proof-of-principle hardware price is currently as expensive as comparable devices already on the market. However, since this price estimation is based on commercially available computers, displays, and other electronics, we expect the price to be reduced significantly. The total prices for the cassette, hardware, and additional equipment are 1.38 EUR/reaction, 0.62 EUR/reaction, and 667.45 EUR/device respectively.

Fig. 10 Price comparison between different versions of AptaVita (based on the stage of development of the company) and the Puremed RDT for vitamin D. The green columns indicate the price of a test cassette for 4 reactions (example of multiplex) and red columns indicate the price per reaction.

Fig. 11 Price comparison between AptaVita hardware with 0%, 50% and 100% margins on the cost price of the prototype and the Hemocue (an unknown margin is already incorporated by the company so no additional margin was calculated for this product) [85]

To cover the expenses made in the early start-up phase, funds must be raised. This can be done through attracting investors, or by applying for funds and innovation competitions. To acquire the former, the product and the company must be pitched to potential investors. The pitch discusses the business model and its potential in the market. To receive feedback on our business model, we collaborated with Florida State University and we had our business model and market analysis reviewed by SkylineDx. We also investigated potential funding opportunities, and found that there are large research funds that we could apply for from, for example, the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) [86]. Furthermore, competitions to gain more attention increase your chances of finding investors and raising funds. In the Netherlands, such competitions include the Philips Innovation Award and Dragons Den [87, 88].

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