Toward point-of-care management of chronic respiratory conditions: Electrochemical sensing of nitrite content in exhaled breath condensate using reduced graphene oxide

OPEN Microsystems & Nanoengineering (2017) 3, 17022; doi:10.1038/micronano.2017.22 www.nature.com/micronano ARTICLE Toward point-of-care management of chronic respiratory conditions: Electrochemical sensing of nitrite content in exhaled breath condensate using reduced graphene oxide Azam Gholizadeh1, Damien Voiry2, Clifford Weisel3, Andrew Gow4, Robert Laumbach3, Howard Kipen3, Manish Chhowalla2 and Mehdi Javanmard1 We present a portable non-invasive approach for measuring indicators of inflammation and oxidative stress in the respiratory tract by quantifying a biomarker in exhaled breath condensate (EBC). We discuss the fabrication and characterization of a miniaturized electrochemical sensor for detecting nitrite content in EBC using reduced graphene oxide. The nitrite content in EBC has been demonstrated to be a promising biomarker of inflammation in the respiratory tract, particularly in asthma. We utilized the unique properties of reduced graphene oxide (rGO); specifically, the material is resilient to corrosion while exhibiting rapid electron transfer with electrolytes, thus allowing for highly sensitive electrochemical detection with minimal fouling. Our rGO sensor was housed in an electrochemical cell fabricated from polydimethyl siloxane (PDMS), which was necessary to analyze small EBC sample volumes. The sensor is capable of detecting nitrite at a low over-potential of 0.7 V with respect to an Ag/AgCl reference electrode. We characterized the performance of the sensors using standard nitrite/buffer solutions, nitrite spiked into EBC, and clinical EBC samples. The sensor demonstrated a sensitivity of 0.21 μA μM− 1 cm − 2 in the range of 20–100 μM and of 0.1 μA μM − 1 cm − 2 in the range of 100–1000 μM nitrite concentration and exhibited a low detection limit of 830 nM in the EBC matrix. To benchmark our platform, we tested our sensors using seven pre-characterized clinical EBC samples with concentrations ranging between 0.14 and 6.5 μM. This enzyme-free and label-free method of detecting biomarkers in EBC can pave the way for the development of portable breath analyzers for diagnosing and managing changes in respiratory inflammation and disease. Keywords: exhaled breath condensate; nitrite; electrochemistry; square wave voltammetry; thin-layer-reduced graphene oxide Microsystems & Nanoengineering (2017) 3, 17022; doi:10.1038/micronano.2017.22; Published online: 22 May 2017 INTRODUCTION Biomarkers have enormous potential utility in assessing chronic inflammation, especially in asthma, which affects ~ 300 million people worldwide. Asthma, which is characterized by variable airway inflammation and air flow obstruction, is an increasingly important global health problem. In the United States alone, ~ 17.7 million adults and 6.3 million children were diagnosed with asthma in 2014 (Ref. 1). Furthermore, the cost of asthma care in the United States was estimated to be $56 billion in 2007. The currently available non-invasive methods for diagnosing and monitoring asthma, i.e., spirometry and the measurement of exhaled nitric oxide, are limited by low sensitivity and the need for expensive and bulky equipment. Moreover, existing tests have a limited ability to characterize the nature and extent of underlying airway inflammation, which is widely variable between individuals2. Measurement of biomarkers in exhaled breath condensate (EBC) can contribute to the molecular phenotyping of asthma, thus enabling targeted treatment and more effective disease management. Given the large and growing burden of asthma, there is an urgent need for improved, minimally invasive methods for the molecular diagnosis and monitoring of asthma. The use of biomarkers in EBC may help to overcome the difficulties associated with obtaining airway tissue and bronchoalveolar lavage samples that have significantly hampered the study of naturally occurring exacerbations of asthma. EBC contains droplets of airway lining fluid (ALF) that are exhaled during normal tidal breathing. In addition to condensed gas-phase compounds, EBC contains non-volatile compounds that originate from ALF, including hydrogen peroxide, nitrite and nitrate, as well as larger molecules such as eicosanoids, proteins, and even nucleic acids3–5. The ability to non-invasively characterize airway tissue by repeated measurements of biomarkers in EBC would be invaluable for studying the time-course of dynamic inflammatory pathways that are involved in asthma exacerbation. Ultimately, EBC biomarkers may contribute to the assessment of different asthma phenotypes and the development of individualized rational approaches to asthma management at the point of care6–9. Recent studies have shown the promise of EBC nitrite for use as a biomarker of both oxidative stress and inflammation in asthma (Figure 1). The primary source of nitrite in the respiratory tract is nitric oxide (NO), which is produced from L-arginine by nitric oxide synthase. In aqueous solution, NO reacts rapidly with reactive 1 Department of Electrical and Computer Engineering, Rutgers University, Piscataway, NJ 08854, USA; 2Department of Material Science and Engineering, Rutgers University, Piscataway, NJ 08854, USA; 3Environmental Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ 08854, USA and 4School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA Correspondence: Mehdi Javanmard () Received: 16 August 2016; revised: 7 December 2016; accepted: 23 December 2016 Graphene oxide for sensing EBC nitrite A Gholizadeh et al 2 Condenser Exhaled breath O2 – O3 NO–2 NO2 +e– Graphene sensor Current (µA) EBC sample 140 120 100 80 60 40 20 0 0 0.2 0.4 0.6 0.8 1 Voltage vs Ag/AgCI Figure 1 An exhaled breath condensate (EBC) sample is collected, and nitrite content is measured electrochemically. oxygen species (ROS) to form more stable nitrogen oxides, such as nitrite (NO2−) and nitrate (NO−3 )10. Increased levels of NO are associated with inflammatory disease states such as asthma, COPD11, and cystic fibrosis12,13. The increased level of exhaled NO in asthma has been suggested to be due to an increased expression of inducible NO synthase (iNOS) in bronchial epithelium14. Given the relative stability of nitrite in EBC and its promise as a biomarker of chronic respiratory inflammation, we developed a miniaturized probe-free/label-free sensor for the detection of nitrite in EBC. Nitrite is typically detected through one of several spectrophotometric methods (Griess reaction) involving fluorimetry, chemiluminescence, or ion chromatography15–28. The detection limit of fluorimetric methods is 0.1 μM. Chemiluminescence has a lower detection limit (in the nM range). Nitrite concentrations in EBC are in the μM range and are compatible with these detection limits. However, despite the low detection limit provided by these methods, EBC samples are usually pretreated to induce the appropriate reaction and/or to eliminate interfering compounds, such (...truncated)