Use of Biosensors in the Detection of Urea Levels. David Dimitroff, MS4 Indiana University School of Medicine
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1 Use of Biosensors in the Detection of Urea Levels David Dimitroff, MS4 Indiana University School of Medicine 84ZM719: Advanced HIT Professor Brent Orndorff January 16, 2017
2 Abstract In the United States, patients with end-stage renal disease (ESRD) are typically maintained on life-saving renal replacement therapies, such as hemodialysis, but these modalities are not without consequences. ESRD patients face considerable morbidity and mortality from their disease and the inadequacy of dialysis to completely replicate physiologic, native kidney function. Biosensors are a nascent technology that could have an important role in determining the adequacy of renal replacement modalities in removing harmful toxins, such as urea, from a patient s blood. A literature review was conducted to investigate trends in these sensing devices over the past decade. As advancements in biosensing devices are made, renal replacement therapies teamed with these devices may provide a substantial increase in the quality of life of ESRD patients. 1
3 On the continuum of chronic kidney disease, end-stage renal disease (ESRD) represents the point at which a patient s kidneys are no longer able to sustain their function in blood purification; in addition, the kidney functions to maintain the homeostasis of many other metabolic, hematological, and immunological parameters, all of which are perturbed in ESRD (Dimitroff, 2016). In the United States, hemodialysis remains the most common method of extracorporeal blood purification in the ESRD population, with 88.2% of all incident cases receiving this modality of treatment (Incidence, 2015). Compared to native kidney function, however, hemodialysis is rudimentary and the substantial morbidity associated with this form of treatment is at least partly due to the buildup of toxins in the bloodstream, such as urea. For these reasons, researchers have sought to develop methods of continuous monitoring of these blood toxins in an effort to improve dialysis adequacy. Over the past decade, advances in nanotechnology have led to the conception of biosensors, which represent a promising tool for real-time quantification of blood analytes, like urea. Here, a literature review from peer-reviewed journals over the past decade will focus on various types of biosensors used in the determination of urea levels, the current state-of-the-art, and some considerations for the future. Biosensors: A Brief Definition Biosensors are devices that incorporate a biological substance, such as an enzyme, attached to a backbone matrix, which ranges from conducting polymers to nanomaterials (Dhawan, 2008). The biological material is selected such that there is an affinity to the analyte to be investigated. Then, this affinity must be exploited by the sensor such that the level of the desired analyte could be quantified. Finally, this level must be transduced to a recorder or monitor for interpretation by the physician or researcher. Currently, dialysis adequacy is measured by trending serum urea and 2
4 creatinine levels. For this reason, these analytes have generally been used as a starting point for using enzyme-based biosensors in the context of determining the quality of blood purification in ESRD patients. Enzyme-Based Urea Biosensors The development of new urea biosensors have largely mirrored the breakthroughs in nanotechnology. Dhawan et al. (2008) described such a device that exploited the advantages of the optical properties of nanotechnological quantum dots (QDs). QDs are a type of nanoparticle that have a role in biosensing due to their luminescent properties. In this case, a metallic backbone of CdSe/ZnS (cadmium selenide/zinc sulfide) provides the photoluminescent solid surface upon which the urease enzyme are conjugated (Duong, 2007). Molecules of urea interact with the affixed urease and are catalyzed to their degradation products. The degree of enzymatic degradation is directly correlated with photoluminescent intensity: using a transducer, changes in this intensity are displayed on a microelectronic display for real-time analysis of the urea concentration (Dhawan, 2008). In vitro studies found that urea concentrations ranging from mm were detectable in systems which controlled for ph a necessary correlate in order to approximate the ph-buffering capacity of human blood (Duong, 2007). This range is very promising, and would only miss patients who are experiencing extraordinary levels of uremia. One particular challenge of QDs is ensuring the stability of photon generation responsible for the changes in light intensity under differential concentrations of urea in a completely aqueous environment, such as blood (Dhawan, 2008). These challenges have prompted other researchers to look at other methods of quantifying changes in urea concentrations, such as utilizing potentiometric measurements. Potentiometric Urea Biosensors 3
5 Potentiometry is an analytical method that measures electrical potential differences between two electrodes. Increases or decreases in potential based on a chemical reaction, such as the hydrolysis of urea, could be recorded and provide a quantitative determination of urea levels in real-time. Much like QD-based biosensors, potentiometric biosensors typically begin with a nanoparticle shell composed of an inorganic metal, to which a biological material is attached. For instance, Nouira et al. (2013) describes nanoparticles of metallic iron oxide with a polyelectrolyte coating, onto which is an immobilized urease enzyme. When placed in aqueous media, this coating becomes a charged surface, and is thereby instrumental in the electron transfer pathway needed to induce electrical potential changes in a system. In the case of urea, its hydrolysis by urease releases ammonium cations (NH4 + ). A potentiometric biosensor could, then, either detect the increase in electron transfer to the ammonium ions, or even in electrical potential changes in the solution due to changes in ph from differential concentrations of ammonium (Nouira, 2013). Using the underlying principles of potentiometry, nanotechnologybased biosensors have been recently created which are able to simultaneously measure multiple analytes using field-effect transistors. Biosensors for Multi-Analyte Detection The use of field effect transistors (FETs) has increased in popularity due to the ability to detect multiple analytes with high sensitivity, highly portable design, and low cost. Similar to a potentiometric device with oppositely charged electrodes, an FET features a source and drain terminal, with an electrically-conducting nanowire channel between them (Karunakaran, 2015). Enzymes or other biomaterials could be immobilized onto the nanowire surface, and enzymesubstrate binding creates an electrical field, ultimately leading to a change in the drain current, proportional to the concentration of analyte. For ESRD patients, simultaneous monitoring of both 4
6 urea and creatinine concentrations would be particularly advantageous for determining adequacy of dialysis. Marchenko et al. described an FET-based biosensor which utilized ph-sensitive field-effect transistors to measure these two analytes. The biosensor was constructed with two separate channels, one of which had affixed urease; the other, the enzyme creatinine deiminase (Marchenko, 2015). Each enzymatic reaction created an electric field, and changes in the drain output signal were recorded to determine analyte concentrations. The group found that these FET-based biosensors had high selectivity for the desired analyte, with rapid response times with data that were consistent with classic, lab measurement techniques used to validate the biosensor results (Marchenko, 2015). Another group used a similar FET setup to simultaneously measure glucose, cholesterol, and urea, with equally promising results (Ahmad, 2015). Future Directions in Biosensors The progress made in the field of biosensors represents a considerable advancement in the quest to reduce the morbidity and mortality associated with ESRD and inadequate renal replacement therapies. To date, in vitro studies show a promising degree of sensitivity in the use of biosensors for the detection of important toxins monitored in ESRD, such as urea and creatinine. To accelerate this progress and bring it more quickly to the bedside, the International Conference on Bio-Sensing Technology was conceived. Now in its fifth year, the conference is organized around themes, and for 2017, two of those include biosensing interfaces and integrated systems (5 th, 2017). The reviewed literature represents just a segment of the various types of structures that biosensing devices can take. As the technology moves closer to being integrated into the care of ESRD patients on a daily basis, the sensitivity, stability, and reliability of the nanomaterial interfaces is coming under more careful scrutiny. New nano-patterned surfaces are being synthesized in an effort to find the safest and most optimal backbone for securing enzymes 5
7 and other biomaterials (5 th, 2017). Also, the integration of biosensors with renal replacement technology, such as a hemodialysis machine, is largely uncharted territory. In such a combination, the benefits of real-time, continuous monitoring of key analytes, such as urea, creatinine, and electrolytes, could be exploited and used to tailor the duration of dialysis treatment. Based on the highly specific data generated from such biosensors, an era of custom dialysate could emerge that would provide optimum blood purification on a patient-to-patient basis. In any case, the future appears bright for improved quality of life for ESRD patients. Conclusion Patients with ESRD are typically started on hemodialysis as a life-saving renal replacement therapy when their kidney function has reached an irreversible level of depressed function. This treatment only crudely approximates the functioning of the healthy kidney, leaving this patient population with significant morbidity and mortality from rising levels of toxins, such as urea, in the blood due to inadequate dialysis quality (Dimitroff, 2017). This review summarized the latest trends and findings in the field of biosensors geared towards the detection of urea levels. These sensors are constructed on the nanoscale with metallic backbones and attached biomaterials, such as enzymes, which are then able to interact with a desired analyte. Depending on the type of biosensor built, the analyte-enzyme interaction could trigger and light or electrical signal, which is then detected and processed by a device recorder for operator-interpretation of the results. Many of the devices built have shown considerable sensitivity and accuracy in detecting and measuring the appropriate analyte, but additional work is required before integrated systems of biosensors and renal replacement modalities are on the market. Biosensors and the continuous, real-time data which they could generate represent an important step forward in improving 6
8 dialysis prescriptions, timing, and adequacy for decreased mortality and increased quality of life in ESRD patients. 7
9 References 5 th International Conference on Bio-Sensing Technology. Ahmad R, Tripathy N, Park J, Hahn Y. A comprehensive biosensor integrated with a ZnO nanorod FET array for selective detection of glucose, cholesterol, and urea. Chem. Commun. 2015;51: Dhawan G, Sumana G, Malhotra B. Recent developments in urea biosensors. Biochemical Engineering Journal Apr;44(1): Dimitroff D. Nanotechnology & microfluidics in wearable dialysis devices. Unpublished Manuscript. Indiana University School of Medicine, Indianapolis, IN. Duong HD, Rhee JI. Use of CdSe/ZnS core-shell quantum dots as energy transfer donors in sensing glucose. Talanta Oct;73(5): Incidence, Prevalence, Patient Characteristics, and Treatment Modalities. In: 2015 Annual Data Report. United States Renal Data System. Available from: Karaunakaran C, Rajkumar R, Bhargava K. Introduction to Biosensors. In: Karunakaran C, Bhargava K, Benjamin R (Eds.), Biosensors and Bioelectronics (p. 37). Waltham, MA: Elsevier. Marchenko SV, Kucherenko IS. Potentiometric biosensor system based on recombinant urease and creatinine deiminase for urea and creatinine determination in blood dialysate and serum. Electroanalysis Apr;27(7): Nouira W, Barhoumi H, Maaref A, Renault NJ, Siadat M. Tailoring of analytical performances of urea biosensors using nanomaterials. Journal of Physics: Conference Series. 2013;416:
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