Bilisense: An Affordable Sensor for Liver Health

Introduction:: Bilirubin (BR) is a heme metabolism product characterized by its strong yellow pigment. In normal physiology, it is conjugated to glucuronic acid in the liver and converted into urobilinogen in the intestines, which is then excreted in feces and urine. Due to this metabolic pathway, it is an effective biomarker for multiple pathologies; it can indicate liver damage, internal bruising and bleeding, or sepsis. Increased serum BR (hyperbilirubinemia) appears as jaundice (a yellowing of the skin), or a darkening of urine. In infants, it must be treated to prevent brain damage and developmental issues. In adults, hyperbilirubinemia is an obvious biomarker for liver disease or damage.

Hyperbilirubinemia is typically first observed as urine, skin, or eye color change, and then is officially diagnosed by a blood test or, in the case of infants, a light meter. However, the colorimetric nature of initial hyperbilirubinemia screening is subjective and significantly less effective for individuals with darker skin color, who are genetically inclined to develop this condition. Additionally, the blood test requires skilled ambulatory care, which makes BR monitoring cost and time-prohibitive.

In this work, we describe the development of a laser-induced graphene electrochemical sensor for the direct and sensitive detection of BR in serum and urine. The sensor does not require specialized skills and can be used at home. It is sensitive to BR in serum and several different drugs with acute liver toxicity and therefore has the potential for personalized drug dosage.


Materials and Methods:: Polyimide adhesive (ULINE, Wisconsin) was placed onto a glass slide and pyrolyzed with an Epilog Laser Mini 30 W laser engraver at 9 % power and 5 % speed to induce graphene working, reference, and counter electrodes. Ag/AgCl ink (Kayaku Advanced Materials, Massachusetts) was stencil-printed onto the working and counter electrode leads to improve conductivity and the reference electrode interface to establish an electrochemical equilibrium. The electrodes were cured at 100 °C for five minutes, then the working area was defined by sealing the electrode with a piece of Kapton tape.

Electrochemical tests were performed by connecting to a Gamry Electrochemical Interface. To demonstrate the function of the laser-induced graphene electrode, cyclic voltammetry was performed with a 5 mM K₃Fe(III)(CN)₆/K₄Fe(II)(CN)₆ (ferri/ferrocyanide) solution, and scan rate studies were performed. The sensor was then evaluated in a solution of BR dissolved in PBS with minimal DMSO. Performance was also evaluated in serum and urine spiked with a known BR concentration and with added ledipasvir (a hepatitis C drug).


Results, Conclusions, and Discussions:: Raman spectroscopy analysis under a 532 nm laser demonstrates the presence of graphene’s characteristic I_2D peak (Figure 1D). We would expect the I_2D/I_G ratio to be large in pristine graphene. Presently, we have successfully engraved graphene onto the polyimide films, but it is not pristine due to inconsistencies in the laser engraving process. We have achieved better I_2D/I_G ratios with other materials, and therefore believe that process optimization will yield better results.

Under cyclic voltammetry, the LIGs demonstrate pairs of current peaks in ferri/ferrocyanide solution that are equal and inverse (Figure 1A). These peaks increase linearly with the square root of the scan rate, and demonstrate a separate linear relationship with solution concentration (Figure 1A, B). These cyclic voltammetry studies confirm that the LIGs have comparable functionality to commercially-produced graphene electrodes, validating their use as a potential bilirubin sensor.

Square wave voltammetry was used to evaluate the LIG as a bilirubin sensor due to (1) its fast scan time compared to differential pulse voltammetry and (2) the inherent correction for capacitive current that is often measured in cyclic voltammetry. Square wave voltammetry studies have demonstrated a bilirubin peak at 0.43 V at BR concentrations below 100 μM (Figure 1C). A three-point calibration curve demonstrates a linear relationship with 0.42399 μA/μM sensitivity.

These studies demonstrate that our novel LIG electrode platform can detect bilirubin at micromolar amounts, which is well within the range of normal physiology (< 20.5 μM), and covers the range of pathophysiology (20.5 – 150 μM). In the future, we will determine the limits of detection and quantification, test the effect of solution pH on signal, and analyze spiked urine and serum samples to simulate real-world analytical performance. Additionally, we will attempt to co-detect bilirubin with ledipasvir in urine to evaluate this sensor’s potential for simultaneous drug and liver panel monitoring.


Acknowledgements (Optional): : The authors would like to that the USC Graduate School and the Alfred E. Mann Department of Biomedical Engineering for their support.


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