Nitric Oxide strip test for the effective and early diagnosis of Asthma Tushar Warrier1; Radha Saraswathy1; S Narmada2, Akella Sivaramakrishna1 1School of Bio-Sciences and Technology, VIT University, Vellore, India 2Nalam Medical Center and Hospital, Vellore, India Abstract Methods and Materials Coating of strip with prepared solution of 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid and n-(1-naphthyl) ethylenedaimine dihydrochloride According to WHO's most recent statistics, around 235 million suffer from asthma globally and it is also the most potent chronic disease among children. The currently used diagnostic techniques such as Peak Flow meter and Spirometry are not always efficient and accurate for the diagnosis of Asthma and thus there is a definite need for more efficient and easier methods for the screening and diagnosis of Asthma. One of the prominent changes that can be observed in asthma is the increase in Nitric Oxide (NO) content in the exhaled breath of the patients (due to eosinophil associated airway inflammation). Values of NO higher than certain threshold values (in both adults and infants) may signal airway inflammation caused by asthma. Based on this notable change in the exhaled NO content in the breath of asthmatics, we have postulated and attempted to design a cost-effective and easy-to-use Nitric Oxide strip that enables the early and efficient detection of Asthma in patients (especially paediatric cases) through a simple colorimetric change. To determine the most ideal set of compounds that can be used for coating the strip for the NO test, we used a set of docking and affinity based studies using tools such as Autodock, Chemdraw Based on the results of the above, a combination of 2 solutions for the detection of Nitric oxide was arrived at viz. 2,2’ – azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and n-(1-naphthyl) ethylenediamine dihydrochloride A solution was made by mixing 17mM stock of 2,2’ – azinobis(3-ethylbenzthiazoline-6-sulfonic acid) with 0.4mM of n-(1-naphthyl) ethylenediamine dihydrochloride, and finally 5 mL of distilled water was added The strip used was either an aluminium or a paper based template The solution of the two chosen solutions was mixed together effectively and the strip was dipped into the solution a couple of times before performing the test Then the patient was made to exhale onto the strip (preferably through the nose) Hold the coated strip in close proximity to the patient’s mouth/nose so as to enable exhalation directly onto the strip Analyse the colour change on the strip and determine the corresponding level of nitric oxide in the exhaled breath Introduction Asthma is a chronic inflammatory disorder of the airways that affects people all over the world Approximately 5% of the world’s population is affected by this deadly disease which encompasses symptoms such as wheezing, chest congestion and difficulty in breathing There are very few confirmatory tests for the diagnosis of Asthma and these tests are usually based on dynamic measurements such as in the case of Spirometry and Peak Flow Meter tests These tests are not always easy to perform and hence the need of the hour in this case is the development of an easy-to-use and cost-effective test for the efficient diagnosis of asthmatics Determine prognosis and further treatment based on the results of the test Fig. 1: Schematic diagram showing the reaction of the components of NO strip that lead to formation of a coloured product Fig. 3: Schematic chart briefly showing various steps in the use of the Nitric Oxide strip test as an effective diagnostic device Results Conclusions The colour of the strip is seen to change to an orange-red colour. This azo dye colour intensity is measured using a spectrophotometer and the range of colour intensity is standardized for different concentrations of nitric oxide The strip was seen to efficiently detect varied concentrations of nitric oxide and showed clearly detectable colour changes above the threshold concentrations of nitric oxide in asthmatics This strip has been effectively simulated and tested under laboratory conditions, and once its utility is substantiated by clinical testing it can become a potent and widely-used diagnostic device The Nitric Oxide strip was developed by the use of two compounds which react in the presence of NO to give an azo dye which can be colourimetrically measured By carrying out this reaction by using different concentrations of NO, a colourimetric chart was established which shows significantly higher colour at concentrations of NO which are deemed to be higher than the threshold limit in asthmatics Thus after proving its validity in a clinical setup, this simple strip test can effectively be used for early diagnosis of asthma Objective To develop an easy-to-use and effective diagnostic strip-based test for early diagnosis of asthmatics; especially in the case of the paediatric and geriatric patients patients Principle Acknowledgements One of the prominent changes that can be observed in this regard is the increase in Nitric Oxide (NO) content in the exhaled breath of the patients Several pro-inflammatory cytokines are upregulated due to the inflammation in the airways and as a result the calcium and calmodulin-independent NO synthases are recruited which causes an increase in the NO content in exhaled breath Threshold values for exhaled breath Nitric oxide are indicated to be around 25 ppb and 35 ppb in infants and adults respectively Thus our hypothesis was to use this increased NO content in exhaled breath as a potent biomarker for enabling the diagnosis of Asthma – by developing a strip based diagnostic device We wish to thank all the patients who took part In this study. We also thank VIT University and Nalam Medical Center and Hospital for providing all the necessary support and facilities for this study. References Patent Application No. 2334/CHE/2013 – Composition, Kit and Method for diagnosing Asthma Dinakar C. Exhaled nitric oxide in pediatric asthma; Current Allergy and Asthma Reports, 2009;9:30 Barnes PJ, et al; Exhaled nitric oxide in pulmonary diseases; Chest, 2010;138;682 Fig. 1: Schematic diagram showing the method of usage for the Nitric Oxide strip test i.e. by exhalation onto the strip