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The Need for Continuous Biomarker Monitoring

Doctors use many different tools to diagnose patients, one of which is the presence and concentration of certain biomarkers.¹ Biomarkers are molecules, like proteins or sugars, that are associated with a particular disease, condition, or biological process. Several ways exist to measure biomarker concentrations, from lateral flow assays to microarrays, but many of these approaches share a weakness: they only show concentrations at one specific point in time.² These biomarker detection methods can also require expensive reagents and take hours to perform - hours that can be crucial for a successful treatment.¹ Not only do doctors need to wait until the results of the test come back, but the results may not even be correct if the patient developed a condition after the tested sample was taken. One solution to eliminate both problems would be to continuously monitor the patient’s biomarker levels. Unfortunately, many of the standard detection methods, like lateral flow assays and blood-culture assays are too expensive and time consuming to use for continuous monitoring. While there are a plethora of ways to constantly monitor physical conditions, like blood pressure and heart rate, there are almost no methods of doing the same for biomarkers.³ Inspired by the need for a fast diagnosis and continuous monitoring of biomarkers, our team created Bio-Spire, a non-invasive aptasensor.

Bio-Spire

Our goal was to create a biosensor that can up-to-date information about the patient’s condition and can be used for a wide range of conditions. Bio-Spire (from “biology” and “perspire”) is a biosensor that continuously monitors the levels of biomarkers in sweat. Unlike blood, sweat is a non-invasive medium to collect, and unlike saliva or urine, biomarkers in sweat can be continuously analyzed so that healthcare professionals can be notified if/when the patient’s biomarker concentrations change dramatically. Levels of biomarkers in blood and sweat are typically correlated, so changes in the amount of biomarkers in sweat are indicative of changes in the blood. A change in the biomarker level in a patients’ sweat can signify a deterioration of the patient’s condition, which is why it is crucial to get that information to a doctor in a timely manner.⁴
Our biosensor uses aptamers to detect biomarkers. Aptamers are strands of single stranded DNA or RNA that can specifically bind to molecules, like proteins in the case of our project.⁵ When the aptamers bind to their target molecules, the biosensor will output the concentration of the biomarkers in an easy to read graph and a message indicating if there is potential sepsis risk so that healthcare professionals can track changes in the concentrations. While the goal for our project is to make a biosensor that is useful for a variety of conditions, we decided to choose one disease to use as a model and proof of concept, so our project is primarily focused on diagnosing sepsis in post-operational patients.

Our Model Disease

Sepsis is a condition characterized by a life-threatening organ dysfunction caused by a dysregulated host response to an infection.⁶ In other words, when the body’s immune response to an infection starts doing more damage to the body than to the infection, the body is in sepsis. Every year, approximately 1.7 million American adults develop sepsis, and 270,000 people die from sepsis.⁷ In fact, one third of all patients who die in American hospitals have sepsis.⁸ In addition, the United States alone spent around $24 billion on treating sepsis in 2013.⁹ As terrible as these statistics are for wealthy countries like the United States, the problem is even more severe in poorer countries. 85% of worldwide sepsis cases occur in low-mid income countries.¹⁰

The most reliable technique for diagnosing sepsis is to make a blood culture. Unfortunately, this takes 24 hours and is often unreliable.¹¹ That’s where Bio-Spire comes in. By taking advantage of the body’s response to the infection, we can track increases in biomarkers associated with inflammation and other symptoms of sepsis. The infection causes a massive increase in the production of proteins (like cytokines)¹². While many of these proteins stay in the patient’s bloodstream, some are exported into their sweat. Once in the sweat, they can be quantified and tracked by our aptasensor. Doctors and nurses can then use this information to monitor and diagnose the patient in a faster, more efficient way- without waiting for cumbersome blood cultures.

Accessibility for All

As our aptasensor diagnoses a prevalent condition like sepsis, it has to be accessible to as many people as possible. This need for accessibility shaped our project by determining which materials would be used to make the biosensor and detect our biomarkers. For instance, we decided to use aptamers instead of antibodies to bind to the biomarkers to make sure that our device would be cost-effective and easy to store. Aptamers are significantly easier to produce than antibodies, making them much less expensive in the long run. While antibodies can require an immune response or animal cells to manufacture them, we can produce billions of aptamers in just a few hours using PCR. Additionally, aptamers are usually more stable than antibodies and have a longer shelf life.¹³ Both of these features are key to making our device as cost-effective and easy to use as possible. You can read more about our work with aptamers here.

The need for accessibility also affected what materials we used to build the sensor itself. We needed to convert biomarker concentration to a readable output, and we eventually decided to use electrical signalling that can be detected by a potentiostat. When aptamers bind to their substrate, they undergo a conformational change that can affect the electrical properties of an electrode, like conductance and resistance. That meant that we needed a conductive, stable, easy to produce material that is sensitive enough to detect minute changes in conductivity and resistance. The best material for this purpose was reduced graphene oxide (rGO). Its conductivity can be regulated by controlling the number of functional groups attached to rGO, and there was precedent for using rGO to detect the concentration of proteins in solution.¹⁴ Even better, rGO is made from graphite, which is a relatively inexpensive material. Unfortunately, the process of making rGO can require some incredibly toxic materials. Fortunately, we were able to use a bacteria called Shewanella oneidensis MR-1 to synthesize rGO (by engineering Shewanella oneidensis MR-1 with better reducing capabilities), making it significantly easier , more sustainable and safer to produce. You can read more about our work with rGO here.

References

Tang, Y.; Qiao, G.; Xu, E.; Xuan, Y.; Liao, M.; Yin, G. Biomarkers for Early Diagnosis, Prognosis, Prediction, and Recurrence Monitoring of Non-Small Cell Lung Cancer. OncoTargets and Therapy 2017, Volume 10, 4527–4534.
Peña-Bahamonde, J.; Nguyen, H. N.; Fanourakis, S. K.; Rodrigues, D. F. Recent Advances in Graphene-Based Biosensor Technology with Applications in Life Sciences. Journal of Nanobiotechnology 2018, 16 (1).
Kim, D.-H.; Paek, S.-H.; Choi, D.-Y.; Lee, M.-K.; Park, J.-N.; Cho, H.-M.; Paek, S.-H. Real-Time Monitoring of Biomarkers in Serum for Early Diagnosis of Target Disease. BioChip Journal 2020, 14 (1), 2–17.
Dai, X.; Okazaki, H.; Hanakawa, Y.; Murakami, M.; Tohyama, M.; Shirakata, Y.; Sayama, K. Eccrine Sweat Contains Il-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal. PLoS ONE 2013, 8 (7).
Irfan, M.; Khan, R. U.; Qu, F. Aptamers for Personalized Therapeutics. Aptamers for Medical Applications 2021, 179–206.
Patki, V. Sepsis Definitions - Changing Perspectives. Journal of Pediatric Critical Care 2018, 5 (4), 26.
Sepsis. National Institute of General Medical Sciences. U.S. Department of Health and Human Services.
(2021, October 5) Clinical information. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention.
Paoli, C. J.; Reynolds, M. A.; Sinha, M.; Gitlin, M.; Crouser, E. Epidemiology and Costs of Sepsis in the United States—an Analysis Based on Timing of Diagnosis and Severity Level*. Critical Care Medicine 2018, 46 (12), 1889–1897.
Sepsis. World Health Organization. World Health Organization.
Lever, A.; Mackenzie, I. Sepsis: Definition, Epidemiology, and Diagnosis. BMJ 2007, 335 (7625), 879–883.
Bozza, F. A.; Salluh, J. I.; Japiassu, A. M.; Soares, M.; Assis, E. F.; Gomes, R. N.; Bozza, M. T.; Castro-Faria-Neto, H. C.; Bozza, P. T. Cytokine Profiles as Markers of Disease Severity in Sepsis: A Multiplex Analysis. Critical Care 2007, 11 (2).
Thiviyanathan, V.; Gorenstein, D. G. Aptamers and the next Generation of Diagnostic Reagents. PROTEOMICS - Clinical Applications 2012, 6 (11-12), 563–573.
Lee, K.; Yoo, Y. K.; Chae, M.-S.; Hwang, K. S.; Lee, J.; Kim, H.; Hur, D.; Lee, J. H. Highly Selective Reduced Graphene Oxide (RGO) Sensor Based on a Peptide Aptamer Receptor for Detecting Explosives. Scientific Reports 2019, 9 (1).

Team:Rochester/Description