CORE Metadata, citation and similar papers at core.ac.uk Provided by MURAL - Maynooth University Research Archive Library Biosensors and Bioelectronics 77 (2016) 505–511 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Creatinine and urea biosensors based on a novel ammonium ion-selective copper-polyaniline nano-composite M. Zhybak a,b, V. Beni b,n, M.Y. Vagin c, E. Dempsey d, A.P.F. Turner b, Y. Korpan a,n a Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 03680 Kyiv, Ukraine b Biosensors & Bioelectronics Centre, IFM, Linköping University, 58183, Sweden c Applied Physics Division, IFM, Linköping University, Linköping, 58183, Sweden d Centre for Research in Electroanalytical Technologies, Department of Science, ITT Dublin, Tallaght, Dublin 24, Ireland article info abstract Article history: The use of a novel ammonium ion-specific copper-polyaniline nano-composite as transducer for hy- Received 22 July 2015 drolase-based biosensors is proposed. In this work, a combination of creatinine deaminase and urease Received in revised form has been chosen as a model system to demonstrate the construction of urea and creatinine biosensors to 29 September 2015 illustrate the principle. Immobilisation of enzymes was shown to be a crucial step in the development of Accepted 3 October 2015 the biosensors; the use of glycerol and lactitol as stabilisers resulted in a significant improvement, Available online 9 October 2015 especially in the case of the creatinine, of the operational stability of the biosensors (from few hours to at Keywords: least 3 days). The developed biosensors exhibited high selectivity towards creatinine and urea. The Creatinine sensitivity was found to be 8573.4 mA MÀ1 cmÀ2 for the creatinine biosensor and Urea À À 112 73.36 mA M 1 cm 2 for the urea biosensor, with apparent Michaelis–Menten constants (K ), Amperometric ammonium detection M,app obtained from the creatinine and urea calibration curves, of 0.163 mM for creatinine deaminase and Copper polyaniline composite 0.139 mM for urease, respectively. The biosensors responded linearly over the concentration range 1– 125 mM, with a limit of detection of 0.5 mM and a response time of 15 s. The performance of the biosensors in a real sample matrix, serum, was evaluated and a good cor- relation with standard spectrophotometric clinical laboratory techniques was found. & 2015 Elsevier B.V. All rights reserved. 1. Introduction this approach is quite complex, time consuming and has limited sensitivity/specificity. Ongoing demand for simple-to-use, cost Chronic kidney disease (CKD) is a common condition with a effective and decentralised diagnostic tools for clinical practice has high risk of death. The National Health and Nutrition Examination significantly boosted the development of alternative methods. In Survey in America has estimated the prevalence of the CKD to be this context, over the past few decades, electrochemical sensors/ about 16% (Haynes and Winearls, 2010). One of the main con- biosensors have been making inroads. Among the electrochemical sequences of CKD is a significant increase in the level of metabolic biosensors developed, potentiometric transduction based on the waste products in the blood, including urea and creatinine, that combination of ion-selective electrodes and ammonium ion-gen- are normally excreted by the kidney (Vaidya et al., 2008). In CKD erating enzymes, is the most common approach used (Magalhaes , patients, urea and creatinine levels can reach up to 10-fold those and Machado, 2002; Osaka et al., 1998; Radomska et al., 2004a , fi recorded in healthy patients, rising from 1.7–8.1 mM (Eggenstein 2004b). The rst example of a potentiometric biosensor for the detection of creatinine was proposed by Meyerhoff and Rechnitz, et al., 1999)to50–70 mM for urea (Walcerz et al., 1998) and from who used creatinine deiminase (CDI) coupled with an ammonia 40–150 mMto1–1.4 mM for creatinine (Ashwood et al., 2006). gas-sensing electrode (Meyerhoff and Rechnitz, 1976). Latter, CDI Consequently, frequent creatinine and urea monitoring, with rapid immobilised in a bovine serum albumin (BSA) (Soldatkin et al., availability of results, can be expected to be of significant benefit 2002a) and PVA/SbQ membrane (Soldatkin et al., 2002b) was used for critically ill patients with remarkable improvement in quality in combination with ion-sensitive field-effect transistors (ISFETs) of life for peritoneal dialysis patients (Udy et al., 2009). for creatinine detection and hemodialysis control (Zinchenko et al., Detection of creatinine dates back to the beginning of the 20th 2012). However, these biosensors suffer from the inherent lim- century when Jaffe method was developed (Jaffe, 1886). However, itations of potentiometry, i.e. vulnerability to interference from other ions present in the sample solutions, relatively slow re- n fl Corresponding authors. sponse times, and high detection limits in biological uids (Lad E-mail addresses: [email protected] (V. Beni), [email protected] (Y. Korpan). et al. ,2008). http://dx.doi.org/10.1016/j.bios.2015.10.009 0956-5663/& 2015 Elsevier B.V. All rights reserved. 506 M. Zhybak et al. / Biosensors and Bioelectronics 77 (2016) 505–511 In order to overcome the limitations of these potentiometric biosensor was between 50 and 250 mM, but it suffered from sig- sensors, several authors have explored the use of multiple en- nificant limitations when applied to biological samples, requiring zymes systems in combination with amperometric detection. One pretreatment with anion exchange separators for the removal of of the first examples of this approach was proposed in a paper by interferants (Rajesh et al. 2005). Tsuchida and Yoda (1983) in which the combination of three en- Polyaniline-Nafion composites have been used to develop am- zymes; creatininase, creatinase and sarcosine oxidase, was used perometric urea biosensors (Cho and Huang, 1998), both on Au for the amperometric detection of creatinine. Electrochemical (Luo and Do, 2004) and Pt support electrodes (Stasyuk et al., 2012). transduction was achieved via the oxidation of the hydrogen Experimental work over the past decade has shown that PANi- peroxide, generated as part of the enzymatic reaction, at a plati- film, deposited on a metallic electrode (Au, Pt), and in combination num electrode. A similar enzymatic reaction sequence was ex- with a cation exchange matrix (Nafion), is highly sensitive to plored by Nguyen et al. (1991), who coupled the three-enzyme ammonium ions (Strehlitz et al., 2000). system with a Clark oxygen electrode. Schneider et al. (1996) and In the current paper, we report the use of a novel ammonium Erlenkotter et al. (2002) reported a poly(carbamoyl)sulfonate ion-selective composite material, based on polyaniline and copper (PCS) hydrogel biosensor that presented significant improvement (Zhybak et al., 2015) for use as a transducer in electrochemical in storage stability (ca. 3 months at 8 C) and dynamic range. The hydrolase-based urea and creatinine biosensors. The experimental use of nano-composite electrodes based, for example, on zinc designs and analytical performance of the biosensors are detailed oxide or iron oxide nanoparticles or carbon nano-tubes, has been together with their application for the determination of creatinine also explored as a way of improving the performance of these and urea in serum samples from patients with CKD. biosensors (Pundir et al. 2013). However, the electrochemical de- tection of oxygen and/or hydrogen peroxide continued to present several limitations in terms of sensitivity and specificity. 2. Material and methods In order to overcome these issues, the use of mediators to- gether with the three-enzyme cascade system was explored. Ra- 2.1. Reagents manavicius (2007) was one of the first to report on the use of a mediator, ferricyanide, for a creatine biosensor. This was despite Creatinine deiminase (EC 3.5.4.21, microbial, Z25 U/mg) was the fact that mediated electron transfer from sarcosine oxidase purchased from Sorachim (Spain). Urease (EC 3.5.1.5, from Cana- had been reported by a several authors earlier on (Taniguchi et al., valia ensiformis (Jack Beans), Z600,000 U/g), urea, creatinine 1988; Turner et al., 1987). However, the dominance of point-of- (anhydrous), hydrochloric acid, copper nitrate, nitric acid, sodium s care analysers (where sensors are required to be re-used) as op- chloride, KH2PO4,Na2HPO4,5%Nafion perfluorinated resin so- posed to home-use devices for this particular analyte has meant lution, glutarealdehyde (GA) (50% w/v aqueous solution), ethanol, that this approach has not found favour to date. This led authors to D-Lactitol monohydrate, glycerol, bovine serum albumin (fraction explore the use of a four enzyme system in which peroxidase V, 96%) were purchased from Sigma-Aldrich (Germany). Aniline (horseradish peroxidase) was added as biocatalyst for hydrogen (99%) was purchased from Sigma-Aldrich (Germany) and redis- peroxide to promote more effective electron transfer (Nieh et al., tilled before use (Important: only freshly prepared aniline should 2013). Unfortunately, when multiple reactions are run in the same be used for electropolymerisation). Ammonium chloride was membrane, optimal conditions for each reaction can often not be purchased from Fluka (Germany). All the chemicals and solvents satisfied and this means that in practice, none of the reactions runs used were of analytical-reagent grade and used without further at its optimum in terms of reaction rate and stability (Ricca et al., purification. 2011). 20 mM phosphate buffer solution (PBS) (prepared by mixing Unlike creatinine biosensors, urea biosensors are commonly and diluting 20 mM disodium hydrogen phosphate, 20 mM po- constructed using only one enzyme, urease (EC 3.5.1.5), as the tassium dihydrogen phosphate and 20 mM sodium chloride) pH biorecognition component, which catalyses the hydrolysis of urea 7.4, was used as the supporting electrolyte during all the electro- to ammonium and bicarbonate ions (Singh et al., 2008).