Team:UofUppsala/Implementation


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Implementation


On this page we give application-oriented ideas on how to implement our cellular biosensor in the real world.

Creating a New Nanobody

NANOFLEX is meant to be adaptable for new applications. Here we present a pipeline to generate a nanobody (sdAb) that fits your target. To know more about the background and techniques check out our Nanobody Guideline.

Nanobody Guideline PDF

Tuberculosis Detection

In order to understand the potential and complications of developing a useful biosensor, we had to choose a target for detection. We wanted to choose a useful target, something that would set hard requirements to fulfill for NANOFLEX. For that purpose, we chose tuberculosis as the case study for our biosensor.

The detection and diagnosis of tuberculosis (TB) is necessary in order to improve health conditions worldwide. It is a widely distributed disease, known and studied for centuries, and there are many protocols and methods already established for dealing with this microbial killer. Developing new technologies that facilitate testing tends to be useful, but it is necessary to understand the health system protocols for designing a diagnostic tool that addresses the current shortcomings. There are many monoclonal antibodies used in TB diagnosis that can be engineered to be used with NANOFLEX (1, 2). We researched and interviewed professionals on TB in order to acquire this knowledge, which you can read more about here (3).

Right now, there are several diagnostic methods widely used in the world. All of them have advantages and disadvantages, but they serve different purposes depending on what needs to be achieved. Some of the diagnostic methods are described below including ideas on how NANOFLEX could be designed.

X-ray

When a patient shows the symptoms of an active TB infection, a cheap and fast method of diagnosis is the use of x-rays in the chest area. This method is used as a complementary method mainly after a bacterial growth test, but it is not conclusive on its own. If the patient has TB their lungs will show the easily recognized granulomas, characteristic of the disease, but it can also show old healed lesions. This test allows only for the detection of active TB and it needs specialized equipment in a hospital (4).

PCR

Reliable PCR diagnostic tools were developed rather recently. It has the massive benefit of allowing for the differentiation of resistant strains of TB, and although the consumables and machines necessary for its performance limit the places where it can be used, it is comparably portable. From what we heard from Nuno Rufino de Sousa, it can even be transported by canoe!

Microbial Culture

The historic golden standard of TB diagnosis is culturing the bacteria from a sample in a selective media and identifying it later using common microbiology techniques. The main problem when using this method is the time it takes to obtain results from it. It can take anywhere from 2-4 weeks to obtain conclusive results, so long in fact that it’s recommended to start the antibiotic treatment before obtaining a diagnosis.

TB Skin Test

The TB Skin Test, also known as the Tuberculin Test, consists of injecting a mass of inactivated TB cells under the skin of a patient. If said patient has been exposed to the bacteria before, the skin will swell up due to the immune response. If the swelling is bigger than a certain threshold, the test is positive. This test is problematic because it cannot distinguish between active and latent TB infection, the patient has to visit the health center twice and it requires heavily trained health workers to perform the test correctly, as the swelling may barely be visible.

IGRA

Interferon Gamma Release Assay (IGRA) consists of taking a blood sample, exposing that sample to TB antigens and measuring the immune response in regards to the amount of interferon gamma in the sample. If the patient was already exposed to TB, the T-cells will produce excess interferon gamma. This test has the massive benefit of using an easily accessible tissue, blood, as a sample rather than sputum. The main problems are that this method doesn’t distinguish between active and latent TB and usually requires heavily specialized equipment for the measurement of interferon gamma, which in urban areas often only is accessible in central laboratories.

Biosensor kits with NANOFLEX

Design 1: Portable Multiplexed Biosensor

NANOFLEX could be defined as a portable multiplexed single-use kit that would allow for fast triage and detection in areas out of reach of current methods. It would be a self-contained device that detects characteristic biomarkers in sputum, blood or urine, with minimal intervention by medical experts whilst giving clear results.

It would serve as a triage system in advance of more specialized methods such as x-rays or a tuberculin skin test, while simultaneously offering more flexibility in transport and use than a PCR due to it not requiring specialized equipment. It could also include small decentralized versions of IGRA, as interferon-gamma detection would be a reasonable target for detection and measurement and to make this useful test widespread. The device could also include other tests for known comorbidities to tuberculosis, such as e.g. HIV (5) or possibly COVID-19.

Design 2: Health Center Benchtop Biosensor

Our second NANOFLEX design is focused on improving the detection methods for health professionals. A laboratory-based kit containing a cellular biosensor can be used to accelerate the detection of mycobacteria in culture from several weeks to just a few days (6). This kit can be transported and commercialized similarly to how bacterial strains are transported today, and the materials for growing new cells are accessible to laboratories worldwide.

References

  1. Kolk, A. H. J., Verstijnen, C. P. H. J., Schöningh, R., Kuijper, S., Peerbooms, P., Rienthong, D., Koanjanartj, S., Polman, K., Maselle, S. Y. and Ho, M. L. (1991). Identification of Mycobacteria by ELISA Using Monoclonal Antibodies. in Rapid Methods and Automation in Microbiology and Immunology, pp. 255-266, Springer, Berlin, Heidelberg.
  2. Wilkins, E. G. L. and Ivanyi, J. (1990). Potential value of serology for diagnosis of extrapulmonary tuberculosis. The Lancet. 336(8716), 641–644.
  3. Centers for Disease Control and Prevention (2020) Tuberculosis. Division of tuberculosis elimination. [online] https://www.cdc.gov/tb/default.htm (Accessed October 25, 2020)
  4. Varaine, F., Rich, M. L., Grouzard, V., Barrera-Cancedda, A. E., Keshavjee, S., Mitnick, C., Mukherjee, J., Peruski, A., Seung, K., Ardizzoni, E., Baert, S., Balkan, S., Day, K., Ducros, P., Ferlazzo, G., Ferreyra, C., Gale, M., Hepple, P., Henkens, M., Hewison, C., Hurtado, N., Jochims, F., Rigal, J., Roy, J., Saranchuk, P., Van Gulik, C. and Ruffinen, C. Z. (2020). Tuberculosis. Médecins Sans Frontières. https://medicalguidelines.msf.org/viewport/TUB/latest/3-7-radiology-20321079.html (Accessed October 25, 2020)
  5. Samuels, J. P., Sood, A., Campbell, J. R., Khan, F. A., and Johnston, J. C. (2018). Comorbidities and treatment outcomes in multidrug resistant tuberculosis: a systematic review and meta-analysis. Scientific reports. 8(1), 1-13.
  6. Verstijnen, C. P. H. J., Schöningh, R., Kuijper, S., Bruins, J., Ketel, R. J. V., Groothuis, D. G., and Kolk, A. H. J. (1989). Rapid identification of cultured Mycobacterium tuberculosis with a panel of monoclonal antibodies in western blot and immunofluorescence. Research in microbiology. 140(8), 653-666.