Creatinine sensitive biosensor based on ISFETs and creatinine deiminase immobilised in BSA membrane
Introduction
In the last decade, there is an increasing demand for simple, selective, accurate and reliable assays for the important metabolites to be used as an indication of health status. Creatinine is the final product of creatine metabolism in mammalian cells. Therefore, it is an important diagnostic substance in biological fluids. Creatinine analysis can be used for the diagnosis of renal, thyroid and muscle function and is very important in treatment with external dialysis. The normal range for serum/plasma creatinine is 35–140 μM [1] and during kidney disfunction it can rise to concentration higher than 1000 μM [2] and its concentration is not very much affected by diet changes.
Most creatinine determinations in clinical biochemical laboratories and almost all the commercially available analysers are based on spectrophotometric detection of Jaffé reaction [3]. However, this reaction is not very specific and many substances such as glucose, pyruvate, acetoacetate, bilirubin, dopamine and other, can interfere [4]. Although some kinetic methods and different pre-treatments of the samples were developed, the accurate determination of creatinine concentration in the biological samples remains a problem.
Enzymatic biosensors seem promising tools for such analysis, and various types of such biosensors for creatinine detection were developed (Table 1). They are: amperometric biosensors using mono and tri-enzyme system [5], [6], [7], [8], [9], [10], [11], [12]; potentiometric creatinine sensor based on ISFET [15], [16] and ion or gas sensitive electrodes [13], [14], [16], [17], [18], [19]; microcalorimetric biosensor based on a pair of silicon microcalorimeters [20], optic sensor [21] and impedimetric one [22], [23]. But as can be seen from Table 1, where a sampling of some existing devices is presented, all these biosensors have some disadvantages such as: low selectivity for amperometric and some potentiometric biosensor; low sensitivity for optic, impedimetric and microcalorimetric sensor; ENFET was not reproducible and stable and needed a reference electrode; some biosensors [5], [10], [14], [15], were with low storage stability. Furthermore, even with good analytical characteristics [16], none was specifically designed for creatinine sensing in dialysis fluids. The bioanalytical system presented in [17], [18] was quite sensitive, selective and stable, but it was not really a biosensor system. The authors used separately immobilised CD in the reactor and ammonium sensitive electrode. In this case complicate and not very cheap system was fabricated and a quite big amount of expensive enzyme preparation was used.
In this investigation we propose to elaborate a very sensitive, selective and stable creatinine biosensor based on differential pair of ISFETs and CD immobilised on the transducer surface. The principle of creatinine sensitive biosensor is based on the following reaction of creatinine hydrolysis catalysed by CD immobilised on the dielectric gate of the pH-sensitive ISFET:which is accompanied by an increase in pH value in enzymatic membrane nearly to the transducer surface. The pH shift induced by the hydrolysis of creatinine and proportional to substrate concentration in analysed solution is registered by ISFET with high sensitivity and accuracy. Potential advantages of ISFETs as transducers for biosensor development include their high sensitivity, the compatibility with integrated circuit technology, possibility for miniaturisation and multisensing implementations and suitability for large scale production at low unit cost [24].
Section snippets
Reagents
Creatinine deiminase (CD) (EC 3.5.4.21) from Microorganism, 41 U mg−1 prot, bovine serum albumin (BSA), glucose and urea were purchased from Sigma. Creatinine, creatine monohydrate, sarcosine were purchased from Fluka. Aqueous solution (25% w/v) of glutaraldehyde (GA) was obtained from Serva. All other chemicals were of analytical grade.
Enzyme immobilisation
A biologically active membrane on the transducer surface was formed by protein cross-linking in saturated GA vapour [25]. Solutions of CD (8% w/v) and BSA (8%
Effect of buffer concentration and enzymatic membrane composition on the sensitivity of creatinine ENFET
Calibration curves for the creatinine sensitive ENFET are presented in Fig. 1. The biosensor demonstrates a rapid response upon creatinine addition (1.5–2 min) and exhibits the dependence of sensitivity and dynamic range on buffer concentration. Creatinine biosensor demonstrates a lower sensitivity and a wider dynamic range when measurements are done at higher buffer capacity. Increase in phosphate buffer concentration from 2.5 to 10 mM results in 4–5-fold decreases in output signal and extends
Conclusions
A quite sensitive, selective and stable creatinine ENFET was developed. The main working characteristics of the developed biosensor such as minimal detection limit, dynamic range and selectivity show that it can be used for a direct creatinine detection in whole blood and/or blood dialysate. Moreover, as the ISFETs are used as transducer for creatinine biosensor elaboration, creatinine sensitive ENFET can be used as monoelement in a multibiosensor development for medical diagnostic and control
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