Background and Motivation


Diabetes is a common disease with multiple complications, which is why it has become an important challenge for world health. According to the World Health Organization (2013), there are around 347 million people around the world with diabetes. On this note, the countries with the greater incidence of diabetes are China, India, United States, Brazil, Russia and Mexico. Since there are no apparent symptoms during the initial states of the disease, early detection and treatment are of great importance to prevent serious complications like heart attack, blindness, kidney failure, lower limb amputation and even death.

In Mexico, in particular, the incidence of diabetes has increased quite notably over the years. This situation has multiple implications for society and for the health system in the country given the negative effects on the quality of life of the patients and their families, as well as the need for a very large economical investment for their proper treatment.

Currently, the glucometer is a widely used device to measure the levels of glucose in the blood of patients with diabetes. However, there are no practical ways of diagnosing other complications that may derive from diabetes. This means that further laboratory testing is needed to verify the presence of said complications after the patient shows symptoms, which takes more time, is more expensive and could result in a late diagnosis with irreversible consequences. This way, our proposal is centered on developing an aptamer-based biosensor that allows the practical and timely diagnosis of one of the irreversible complications that result from diabetes: the extensive damage to the retina known as retinopathy.



Project Description


The advanced glycation end-products (AGEs) are a group of compounds that have been implicated in diabetes related complications. Because of their complex and heterogenous nature, it has not been proved if they are the cause or the consequence of those complications. However, in vitro work has shown AGEs to be part of the complex interactions within oxidative stress and the accelerated vascular damage that occurs particularly in patients with atherosclerosis and diabetes. Despite their complexity and distribution in different pathologies, the known AGEs produce the formation of covalent crosslinks between proteins, which are thought to be one of the central underlying processes that explain the damage they cause. Furthermore, most research on AGE has centred on diabetes mellitus and its complications, since diabetes is associated with premature atherosclerosis. For diabetic patients this relates to complications like neuropathy, retinopathy and lens disorders.

One of the main problems is that currently there is no universally accepted method to detect AGE, which is why the timely diagnosis of derived complications is limited. This way, our project focuses on the solution of this situation. To do so, the goal is to develop a practical, non-invasive and economic aptamer-based electrochemical biosensor (electrochemical aptasensor), which allows to quantify glycated albumin. The chosen DNA aptamer is AGE-HAS which can bind with high affinity and specificity. An aptamer was determined as the best option for the biological component in the biosensor because of the many advantages they have over the commonly used antibodies, including their high affinity and specificity, thermal stability, cost-effectiveness, great accuracy and reproducibility of their production, low immunogenicity and wide variety of targets.



Overview


This project aims to develop an electrochemical analysis system through the use of and aptamer and the glycated albumin as a biomarker. In order to achieve this, several preliminary tests are carried on for the electrochemical analysis system. As well, other characteristics of the biosensor, such as specificity, high affinity, electrochemical characterization of the electrode and proper functionalization of the aptamer based electrode are tested through performing controls in order to verify that these properties are successfully accomplished.

We decided to develop this project due to the fact that there is not available a general method that may be able to detect AGEs at an early stage before they begin causing damage. Another reason is that the current methods available represent an expenditure that can not be paid by many people.

The effectivity of the biosensor can be achieved through the performance of calibration curves with different concentrations of the analyte, so that the results obtained could be used in order to set the limits of detection and quantification desired.



Objectives


  • Mounting an electrochemical analysis system using the aptamer and glycated albumin as a biomarker.
  • Conducting preliminary tests for the electrochemical analysis system.
  • Proper functionalization of the aptamer based electrode and electrochemical characterization of the electrode.
  • Performing controls to verify the specificity of the biosensor.
  • Performing calibration curves with different concentrations of analyte to establish the limits of detection and quantification of the biosensor.



    • Materials, equipment, and methods


    Materials

  • Anti-glycated albumin aptamer. Synthesized at Sigma-Aldrich with HPLC purification (Yuichiro Higashimoto., et al, 2007)
  • Glycated albumin.
  • N-acetylcysteamine (NAC).
  • Isopropanol (C3H8O).
  • Fe3+/Fe2+ solution. 1.0 mM K4Fe(CN)6/K3Fe(CN)6.
  • Potassium hydroxide solution 0.5 M (KOH).
  • Milli-Q water 18.2 MΩ•cm.
  • Phosphate Buffered Saline (PBS) pH 7.4, 10 mM (37 mM NaCl, 2.7 mM KCl, 10mM KH2PO4).
  • Screen printed gold electrode (DS220AT) acquired with Dropsens’ company.
  • 5% Sulfuric acid solution (H2SO4).

    • Equipment

  • Analytical balance.
  • Graduated cylinder of 10 ml.
  • Micropipette of 20 - 200 μl.
  • Micropipette of 10 - μl.
  • Autolab Potentiostat PGSTAT204 with modul FRA32M.
  • Teflon cell for modification of the working electrode.

    • Methods


  • Electrochemical Impedance Spectroscopy (IES).

    • Electrical resistance is the ability of a circuit element to resist the flow of electrical current. Ohm's law defines resistance in terms of the ratio between voltage, E, and current I. Nevertheless, its use is limited to only the ideal resistor. However, the real world contains circuit elements that exhibit much more complex behavior that force us to include the term impedance. Impedance is a measure of the ability of a circuit to resist the flow of electrical current, but unlike resistance, it is not limited by the previously mentioned properties.

    Electrochemical impedance is usually measured by applying an AC potential (potential or current) of fixed frequency, to an electrochemical cell and then measuring the current through the cell. This response is then measured and the impedance is computed at each frequency. Finally, the results are plotted and analyzed. For the present project we will be using a potentiostat AUTOLAB which will allow us to receive all the measures at different frequencies (Gamry, 2016).


  • Cyclic voltammetry.
    •

    Cyclic voltammetry has become a popular tool in the last fifteen years for studyingelectrochemwal reactions. The voltage applied to the"working"electrode is scanned linearly from an initial value (Ei) to a predetermined limit (El), known as the switching potential, where the direction of the scan is reversed. .The operator can halt the scan anywhere or let the instrument cycle between E1 and some other preselected value, E2.The current response is plotted as a function of the applied potential. (Mabbott, G. A.1983).


  • Square wave voltammetry (SWV).
    •

    This technique capitalizes on the present revolution in electronics, is a complex but powerful technique that required the power and flexibility of the minicomputer for its development and modern microprocessors for its commercial implementation.

    The square wave voltammetric waveform of combines a large-amplitude square wave modulation with a staircase waveform. The resulting net current (Δι,), a true differential signal, can be obtained at high effective scan rates. The peak-shaped voltammograms obtained display excellent sensitivity and rejection of background currents. The main features of the voltammetric response are best illustrated by considering a simple, reversible redox system (Ramaley, L.1969).




    Notebook


    Link to download the laboratory notebook.



    Results and Discussion


    Electrical resistance is the ability of a circuit element to resist the flow of electrical current. Ohm's law defines resistance in terms of the ratio between voltage, E, and current I. Nevertheless, its use is limited to only the ideal resistor. However, the real world contains circuit elements that exhibit much more complex behavior that force us to include the term impedance. Impedance is a measure of the ability of a circuit to resist the flow of electrical current, but unlike resistance, it is not limited by the previously mentioned properties.

    For the electrode with the NAC treatment (from the previous session), when the graphs of figures 1.a, 1.b and 1.c were analyzed, we could observe that the height of the wave in the impedance test was greatly diminished. This means that the NAC was attached to the electrode, but since the resistance appears to be too big, it can be concluded that this electrode was blocked excessively. As NAC is a blocking agent, the objective is to block the gold surface, but still leave room for the aptamer to attach to it in the spaces left unobstructed by the NAC. This will help with the specificity of the biosensor.
    Figure 1.a, Figure 1.b and Figure 1.c with probe Ferri/Ferro and PBS pH 7.4 Green: Bare gold electrode. Blue: Electrode after modification with Aptamer-NAC. Modification with Aptamer-NAC (10 uM for 16 hr and 1 mM NAC for 3 hr).

    The reason why the NAC treatment ended up blocking the electrode in an excessive way is because the treatment was realized in 16 hours, for the treatment to prevent an excessive blocking it is recommended to leave the treatment for much less than 16 hours.

    There were some errors during the process of cleaning of the electrode, since instead of running the cyclic voltammetry we run the dropsens without pretreatment. So the first electrode was burned. It is also important to emphasize that the techniques used on the experiment are to check how clean the electrode is.

    After repeating all the cleaning process (with a new electrode). The cyclic voltammetry gave an acceptable space between pikes that was 0.083008. As expected on the second technique which was OSWV, both graphs were one above another, which showed that the electrode was clean.

    Also, the non-glycated albumin serves as a negative control. When the tests were performed, the interaction with the aptamer and this protein was quite weak, which can be inferred by looking at the graphs. If we compare the ones obtained with the glycated albumin and the non-glycated albumin, the change in the signal of the glycated one was notably bigger than the one observed with the non-glycated one. This change was inverse, for the glycated albumin the height of the curve is diminished in great measure, while for the non-glycated albumin, the height of the curve increased a little. This validates our system’s specificity, since the aptamer we are using is supposed to interact only with glycated albumin (which is a biomarker for diabetic retinopathy).
    Figure 4.1. Control test, non glycated albumin.

    Figure 4.2. Functionality test, glycated albumin.

    We obtained a calibration curve of OSWV (see figure 3) where it shows that the electrode saturates when the concentration of the glycated albumin reaches approximately 6 ng/ml. We are also missing more points in the lower concentrations to prove that it has a linear behaviour. Figure 3. Calibration curve with voltammetry square wave. Electrode modification with Aptamere-NAC (10 uMduring 16 hours y 1 mM NAC while 2 hours).

    Figure 5. Calibration curve with voltammetry square wave. Azul: 2ng/ml AG. Red: 4 ng/ml AG. Green: 6 ng/ml AG. Purple: 8 ng/ml AG.Electrode modification with Aptamer-NAC(10 uM during 16 hours y 1 mM NAC during 2 hours)

    In figure 5 we can see the calibration curves (OSWV) from the electrodes with different treatments. The blue line was with 2 ng/ml glycated albumin, the red line with 4 ng/ml glycated albumin, the green one with 6 ng/ml glycated albumin and the purple one with 8 ng/ml glycated albumin. Here we can see that the peak heights of the green line and the purple one are one over the other which means that it is saturated at those concentrations.

    Figure 6. Calibration curve of spectroscopy of impedance. Blue: 2ng/ml AG. Red: 4 ng/ml AG. Green: 6 ng/ml AG. Purple: 8 ng/ml AG. Modification of electrode with Aptamer-NAC (10 uM for 16 hr and 1 mM NAC for 2 hr).

    Figure 7. Calibration curve of spectroscopy of impedance. Modification of electrode with Aptamer-NAC (10 uM for 16 hr and 1 mM NAC for 2 hr).



    Conclusions


    Overall, the project objectives were reached. An electrochemical analytic system for Glycated Albumin was developed. Electrochemical Impedance Spectroscopy (EIS), Cyclic voltammetry (CV) and Square wave voltammetry (SWV) were conducted correctly to characterize the system and verify its proper functionalization. According with the tests performed, Glycated Albumine produced an electrochemical signal. Also, a significant difference in signal was produced for Glycated Albumin in comparison with Non-Glycated Albumin, which suggests that the system is specific for the molecule of interest. The calibration curves provide information that the system can be useful for analytical purposes. However, it is important to clarify that the project is in research and development phase, so the obtained prototype requieres improvents in design and further testing. Experiments with real samples (human blood and saliva) are required in order to determine the reliability of the system.



    Future applications

  • Replace the gold electrodes with glassy carbon ones in order to reduce costs.
  • Industrialize the biosensor manufacturing process in order to obtain massive production of the analytical device.
  • Carry out the project in blood samples instead of artificial samples.
  • Adapt the electrochemical system to quantify other biomarkers involved in diabetes and its complications.
  • Optimize the electrode functionalization in order to get bigger ranges of quantification.
  • It is important to spread the idea of prevention. In Mexico, people do not take actions unless they have symptoms of disease. Our purpose is to improve diabetes prevention campaigns, allowing people to be conscious and responsible for their health. Our main goal in a long term is to generate a commercial biosensor device capable of improving the early detection of diabetic retinopathy in the mexican population.



    • References


  • Hernández-Ávila, M., Gutiérrez, J.P. & Reynoso-Noverón, N. (2013). Diabetes mellitus en México. El estado de la epidemia. Salud Pública de México , 55(2), 129-136. Retrieved from: http://www.scielosp.org/pdf/smp/v55s2/v55s2a9.pdf
  • Singh, R., Barden, A., Mori, T. & Beilin, L. (2001). Advanced glycation end-products: a review. Diabetologia, 44, 129-146.
  • Song, K., Lee, S. & Ban, C. (2012). Review: Aptamers and Their Biological Applications. Sensors, 2012(12), 612-631.
  • Higashimoto Y, Yamagishi S, Nakamura K, Matsui T, Takeuchi M, Noguchi M, Inoue H. (2007). In vitro selection of DNA aptamers that block toxic effects of AGE on cultured retinal pericytes. Microvascular Research 74 (2007) 65-69. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/17493639
  • GAMRY Instruments. Basics of Electrochemical Impedance Spectroscopy. Retrieved from: http://www.gamry.com/application-notes/EIS/basics-of-electrochemical-impedance-spectroscopy/
  • GAMRY Instruments. Introduction to Electrochemical Impedance Spectroscopy. Retrieved from: http://www.gamry.com/assets/Uploads/Basics-of-Electrochemical-Impedance-Spectroscopy.pdf
  • Mohd, J., Inho, S., Mohd, N., Uzma, G. (2015). Recent Advances in Detection of AGEs: Immunochemical, Bioanalytical and Biochemical Approaches.
  • Mabbott, G. A. (1983). An introduction to cyclic voltammetry. J. Chem. Educ, 60(9), 697.
  • Ramaley, L., & Krause Jr, M. S. (1969). Theory of square wave voltammetry. Analytical Chemistry, 41(11), 1362-1365.