The use of a novel ammonium ion-specific copper-polyaniline nano-composite as transducer for hydrolase-based biosensors is proposed. In this work, a combination of creatinine deaminase and urease has been chosen as a model system to demonstrate the construction of urea and creatinine biosensors to illustrate the principle. Immobilisation of enzymes was shown to be a crucial step in the development of the biosensors; the use of glycerol and lactitol as stabilisers resulted in a significant improvement, especially in the case of the creatinine, of the operational stability of the biosensors (from few hours to at least 3 days). The developed biosensors exhibited high selectivity towards creatinine and urea. The sensitivity was found to be 85 ± 3.4 mAM(-1)cm(-2) for the creatinine biosensor and 112 ± 3.36 mAM(-1)cm(-2) for the urea biosensor, with apparent Michaelis-Menten constants (KM,app), obtained from the creatinine and urea calibration curves, of 0.163 mM for creatinine deaminase and 0.139 mM for urease, respectively. The biosensors responded linearly over the concentration range 1-125 µM, with a limit of detection of 0.5 µM and a response time of 15s. The performance of the biosensors in a real sample matrix, serum, was evaluated and a good correlation with standard spectrophotometric clinical laboratory techniques was found.

[c] 1IntroductionThed eterminationo fc holinec ontent in fooda nd biological fluids is an important analyticalt ask. Cholinei s indeed indispensable for the metabolism of mammals.I t takes part in the regulation of the insulin level and transportation of the fats in the liver as wella si nt he cell membrane construction. Cholinei ss ynthesized by the organism but additional quantities may be taken with infant food, vitamin formulasa nd sport drinks in order to avoid occurrence of deficiencyd iseases [1].M oreover, the choline determination has been reported for the diagnosis of Parkinson and Alzheimer diseases [2,3].Several methods have been developed for the choline determination, including the couplede nzymatic-spectrophotometric method [4],l iquid chromatography [ 5],e lectrochemi-and chemi-luminescence [6,7].T he actual development of personal healthc are leads to the demand for cheap and reliable sensors,e lectrochemical biosensor being one of the mostr elevant approaches [8].T he enzymatic conversion of choline to betaine aldehyde by choline oxidase (ChOx)l eads to hydrogen peroxide that can be electrochemically detected. An umber of choline electrochemical biosensors have been proposed by the immobilization of ChOx ontoe lectrode surfaces,i ncluding carbon paste electrodes [9,10],e ncapsulation in conducting [11],n onconducting[ 12] and redoxp olymers [13],i n ZnO 2 -matrix [14] using layer-by-layer assembly [3,[15][16][17] or glutaraldehyde coupling [ 2,18,19].D espite this variety of works,only afew of them have described their application as the choline determination in eggs,p asta, milk, dietetic meal, baby food [18,20] and biological fluids [10].ChOx hasa lsob eeni mmobilizedi ns ol-gelt hinf ilms in association with carbon nanotubesa nd metalnanoparticles [21,22]and showsgoodperformance forthe determination of phosphatidyl cholinei ns erum samples [ 21]. Good longterm operationals tability forimmobilized ChOx canbeattributed to theh ybrids ilicam aterials obtained by thes olgelt echnology.T hese materialsare indeed theidealm atrix fore ncapsulating biomolecules whilep reservingt heir activity [23][24][25][26][27]. In principle, thel ow-temperature sol-gel synthesisp rovidess ilicap olymerc hainsf ormation around thee nzymem oleculet hatc an keeps tericc onfiguration of thep rotein intact [26,27]. This givest he possibilityt o obtain aw ider ange of sensitivee lementso fa mperometric sensors basedonsol-gel modified electrodes.Hybrid sol-gel silica are most usually depositedo nelectrode surface by drop-coating the startings ol containing the protein(s), the silica monomer(s) and eventuallys ome additives [ 28].A nother approachh as been proposed reAbstract:Amediator-freec holine biosensor was developed using the electrochemically assisted sol-gel deposition on gold screen-printed electrodes.T he addition of 12 mM of cationic surfactant CTAB in silica sol allowed enhancing the stability of the sensor. Them odified electrode demonstrated catalytica ctivity and stable amperometric response to choline ...

We describe a composite material for use in electrochemical oxygen reduction. A screenprinted electrode (SPE) was consecutively modified with electrodeposited copper, a Nafion membrane and electropolymerised polyaniline (PANi) to give an electrocatalytic composite of type PANi/Nafion/Cu2O/SPE that displays good electrical conductivity at neutral pH values. It is found that the presence of ammonia causes complex formation with Cu(I), and this causes electroreduction of oxygen to result in an increased cathodic current. The finding was applied to the quantification of ammonium ions in the 1 to 1000 µM concentration range by amperometry at -0.45 V (vs. Ag/AgCl). This Faradaic phenomenon offers the advantage of direct voltammetric detection, one of the lowest known limits of detection (0.5 µM), and high sensitivity (250 mA•M -1 •cm -2 ). It was applied to the determination of ammonium ion in human serum where it compared well with the photometric routine approach for clinical analysis using glutamate dehydrogenase.