Determination of creatinine with a sensor based on immobilized glutamate dehydrogenase and creatinine deiminase

Glutamate dehydrogenase and creatinine deiminase are immobilized by adsorption on wet poly(vinyl chloride) membranes. Creatinine is determined by a sensor consisting of the two membranes placed over an ammonia-sensing electrode. Endogenous ammonia is removed as it passes through the glutamate dehydrogenase layer. Creatinine (1–50 mg dl^?1) is converted to ammonia in the inner creatinine deiminase layer and is detected by the ammonia electrode. The assay requires 3 min, the minimum detectable concentration is 1 mg dl^?1 at pH 8.5, and the precision is ca. 5%. Endogenous ammonia can be tolerated up to 2 × 10^?4 M.     

Determination of creatinine in undiluted blood serum enzymatic flow injection analysis with optosensing

A specific enzymatic assay for creatinine in undiluted serum samples is described, exploiting the generation of ammonia from creatinine by immobilized creatinine iminohydrolase. The ammonia produced is separated from the sample matrix by gas diffusion into an acceptor stream containing a pH-sensitive indicator. The creatinine content is quantified by monitoring the resulting colour change of the indicator by means of reflectance measurement via optical fibers, the hydrophobic gas-permeable membrane serving as a diffuse reflector. Two approaches are used to overcome the interference caused by endogenous ammonia. The first is based on enzymatic abatement of endogenous ammonia by immobilized glutamate dehydrogenase. In the second, preferable, approach, endogenous ammonia, itself a parameter of clinical interest, is measured separately prior to the enzymatic degradation by creatinine iminohydrolase. Each assay requires only 30 μl of sample solution, and the sampling frequency is 60 h^?1. The relative standard deviation is approximately 3%.     

Ammonia abatement in an enzymatic flow system for the determination of creatinine in blood sera and urine

The abatement of ammonia in standard solutions, and in human blood and urine samples is achieved by adding suitable amounts of NADPH and α-ketoglutarate to the sample and passing it through a 2-m nylon tube with glutamate dehydrogenase immobilized on the inner wall. The procedure provides removal of 98% of the ammonia (1–5 × 10^?4 M) in the original sample in 50 s. The abatement of ammonia permits the use of an ammonia probe coupled with an immobilized degradative enzyme for the determination of creatinine. Creatinine was determined in clinical blood and urine samples by first removing the ammonia from the sample and then cleaving the creatinine to N-methylhydantoin and ammonia with immobilized creatininase. Only 200 μl of sample is needed and the entire process is conducted in a single flow stream.     

Ammonium ion sensor based on immobilized nitrifying bacteria and a cation-exchange membrane

The ammonium ion sensor is based on nitrifying bacteria isolated from activated sludge. The sensor comprises a cation-exchange membrane, an alkaline solution layer (pH 10), a gas-permeable membrane, an immobilized microbial membrane, and an oxygen electrode. This novel combination provides accurate amperometric determinations of ammonium ions in aqueous solutions within 7 min in the range 10^-4– 4.5 × 10^-2 M. Volatile amines or other ions do not interfere. The relative error is within 4% and the sensor can be used continually for more than 10 days.     

An enzyme immunosensor for the electrochemical determination of the tumor antigen α-fetoprotein

A specific sensor for a tumor antigen, α-fetoprotein (AFP) can be prepared from a membrane with immobilized antibody and an oxygen probe with a permeable teflon membrane. Anti-AFP antibody is covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The sensor is applied to enzyme immunoassay based on competitive antigen-antibody reaction with catalase-labelled antigen. After competitive binding of free and catalase-labelled AFP, the sensor is examined for catalase activity by amperometric measurement after addition of hydrogen peroxide. AFP can be determined in the range 10^-11–10^-8 g ml^-1.     

Array of potentiometric sensors for the analysis of creatinine in urine samples

The development of potentiometric biosensors for creatinine based on creatinine iminohydrolase (E.C. 3.5.4.21) immobilized on chitosan membranes coupled to a nonactin based ammonium ion selective electrode is described. The response characteristics of three types of biosensors with the enzyme immobilized by three different procedures were evaluated. The biosensors with better response characteristics were obtained by coupling the ammonium ion selective electrodes to chitosan membranes with the enzyme immobilized by adsorption. The linear response range of these biosensors to creatinine was 10(-4) to 10(-2) M, the response time was between 30 and 60 s, they showed an operational lifetime of 44 days and the slope of the response to creatinine in the first day varied between 50 and 52 mV decade-1. An array of six potentiometric sensors, constituted by two creatinine biosensors and four ion selective electrodes for potassium, sodium, ammonium and calcium was calibrated and a multivariate model based on PLS1 for the response to creatinine was obtained and validated. The array was used for the analysis of creatinine in urine samples and the results were compared with the results of a clinical analysis laboratory, based on the Jaffé reaction.     

Continuous-flow enzymatic determination of creatinine with improved on-line removal of endogenous ammonia

A new continuous-flow automated enzymatic method suitable for the direct determination of creatinine in physiological samples is described. The proposed system utilizes an on-line gas predialysis unit in conjuction with a flow-through enzyme reactor coil and a potentiometric ammonia detector. The enzyme reactor contains immobilized creatinine iminohydrolase (EC 3.5.4.21) which converts creatinine to ammonia and N-methylhydantoin. Ammonia liberated from this reaction is detected downstream with the membrane electrode-based detector. The novel gas predialysis unit effectively removes >99.8% of endogenous ammonia (up to 1 mM) present in the sample. Thus, final peak potentials recorded by the electrode detector are directly proportional to the logarithm of creatinine concentrations present. The method is shown to be precise (<3%), selective, and capable of accurately determining creatinine in serum and urine samples containing abnormally high endogenous ammonia levels. Determinations of creatinine in serum samples (n = 30) using this new method correlate well with an existing Technicon AutoAnalyzer colorimetric method (r = 0.996).     

Amperometric determination of glucose in undiluted food samples

Glucose oxidase is immobilized onto a cellulose acetate membrane by glutaraldehyde linkage, and the membrane is used to cover the platinum electrode of a hydrogen peroxide sensor. A silanized polycarbonate membrane then covers the enzyme layer, and extends the linear calibration range to higher concentrations. The sensor, when incorporated into a flow-injection system, allows the determination of glucose at levels up to 1 M in soft drinks at a rate of 60 samples h^?1 without sample dilution.     

New potentiometric sensors for creatinine

Three main types of creatinine potentiometric membrane sensors are described. They are based on the use of dibenzo-30-crown-10 (DB30C10) with potassium tetrakis(p-chlorophenyl)borate type (I), dibenzo-30-crown-10 alone type (II), and potassium tetrakis(p-chlorophenyl)borate alone type (III), incorporating in poly(vinyl chloride) matrix membrane plasticized with either o-nitrophenyl octyl ether or dioctylphthalate. The sensors are used for quantification of creatinine after soaking the membranes in 0.1 M creatinine solution for 2 days. The sensors show almost the same potentiometric response characteristics. Sensor type (I) exhibits Nernstian responses over a concentration range of 5.0 × 10⁻⁵ mol l⁻¹-1.0 × 10⁻² mol l⁻¹ creatinine with cationic slopes of 59.5 ± 0.1 and 60 ± 0.2 mV decade⁻¹ and detection limits of 1.1 × 10⁻⁵ mol l⁻¹ and 8 × 10⁻⁶ mol l⁻¹ creatinine, over the pH range of 3.5-6.5 and 3.5-7.0, for o-NPOE and DOP solvent mediators, respectively. Sensor type (II) displays Nernstian responses over a concentration range of 6.0 × 10⁻⁵ mol l⁻¹-1.0 × 10⁻² mol l⁻¹ creatinine with cationic slopes of 60.0 ± 0.1 and 65.0 ± 0.2 mV decade⁻¹ and detection limits of 1.5 × 10⁻⁵ mol l⁻¹ and 1.4 × 10⁻⁵ mol l⁻¹ creatinine over the pH range of 2.6-6.2 and 2.5-6.0, for o-NPOE and DOP solvent mediators, respectively. Sensor type (III) shows Nernstian responses over a concentration range of 7.0 × 10⁻⁵ mol l⁻¹-1.0 × 10⁻² mol l⁻¹ creatinine with cationic slopes of 60 ± 0.1 and 62.0 ± 0.2 mV decade⁻¹ and detection limits of 2.7 × 10⁻⁵ mol l⁻¹ and 2.0 × 10⁻⁵ mol l⁻¹ creatinine over the pH range of 2.5-6.0, for o-NPOE and DOP solvent mediators, respectively. The response times of the sensors for 10⁻³ mol l⁻¹ creatinine solution are instantaneous (4-10 s). The sensors show long-term stability with life span of ∼6 months. The sensors are used for determination of serum creatinine of rats (Rattus Norvigicus) with mean R.S.D. of 2.62%, and the results agreed well with the Jaffe kinetic method.     

Determination of salbutamol using R-phycoerythrin immobilized on eggshell membrane surface as a fluorescence probe

A fluorescence sensor was fabricated using R-phycoerythrin (R-PE) immobilized on eggshell membrane as the fluorescence probe, and salbutamol was determined based on the decrease in fluorescence intensity of R-phycoerythrin. The scanning electron and fluorescence micrographs showed the microstructure of the eggshell membrane and indicated that the R-PE was successfully immobilized on the eggshell membrane surface. The effects of some experimental parameters on the response of the biosensor were investigated in detail. The fluorescence sensor has a linear response to salbutamol concentrations ranging from 5.00 to 100 ng mL⁻¹. The detection limit for the salbutamol is 3.50 ng mL⁻¹ (S/N = 3). The reproducibility of fabricating the biosensors using six different membranes was good with a relative standard deviation (RSD) of 3.28%. The fluorescence sensor showed extremely good stability with a shelf life of at least 50 days and reversible response to salbutamol. Some common potential interferents showed little effect on the response of the salbutamol fluorescence sensor. The proposed method was successfully applied to the determination of the salbutamol in urine samples.     

Determination of creatinine in clinical samples with a creatininase reactor and an ammonia probe

A commercial nylon coil with immobilized creatininase in conjunction with a potentiometric ammonia probe is used in a continuous flow apparatus for the determination of creatinine in blood plasma and urine. The analytical characteristics of the coil are evaluated and its specific enzymatic activity is calculated. The determination of creatinine in plasma is viable provided that samples are processed quickly after they have been taken and that plasma ammonia is evaluated just before the creatinine determination. Analyses of urine require prior separation of ammonia. For plasma, 40 samples/hour can be processed with confidence. The precision of the procedure depends strongly on the plasma ammonia level and is about 5% for normal conditions.     

Alcohol-FET sensor based on a complex cell membrane enzyme system

An alcohol -FET sensor was developed by use of a complex enzyme system in a cell membrane and an ion-sensitive field effect transistor (ISFET). The cell membrane of Gluconobacter suboxydans IFO 12528, which converts ethanol to acetic acid, was immobilized on the gate of an ISFET with calcium alginate gel coated with nitrocellulose. This ISFET (1), a reference ISFET without the cell membrane (ISFET 2) and an Ag/AgCl reference electrode were placed in 5 mM Trismalate buffer (pH 5.5, 25°C), and the differential output between ISFETS 1 and 2 was measured. The output of the sensor was stabilized by adding pyrroloquinoline quinone. The response time was ca. 10 min., and there was a linear relationship between the differential output voltage and the ethanol concentration up to 20 mg l^?1. The output of the sensor was stable for 40 h below 30°C. The sensor responded to ethanol, propan- 1-ol and butan- 1-ol, but not to methanol, propan-2-ol and butan-2-ol. The sensor was used to determine blood ethanol.     

Optical sensor for berberine utilizing its intrinsic fluorescence enhanced by the formation of inclusion complex with butylated-β-cyclodextrin

An optical sensor for berberine, the basic ingredient of the widely used traditional Chinese medicine Coptis Chinensis, based on its intrinsic fluorescence enhanced by butylated-β-cyclodextrin (HDB-β-CD) immobilized in plasticized poly(vinyl chloride) (PVC) membrane has been developed. The drastic enhancement of fluorescence intensity of berberine was attributed to the formation of an inclusion complex between HDB-β-CD and berberine, which has been utilized as the basis of the fabrication of a berberine-sensitive fluorescence sensor. The proposed sensor was quite distinct from those fluorescent sensors for berberine reported so far which relied upon quenching the fluorescence of the sensing reagent immobilized on membrane by berberine. The response mechanism of optode membrane was discussed in detail from the view of molecular dynamics and the optimum steric configuration of the inclusion complex was presented by molecular dynamics simulation. The analytical performance characteristics of the proposed berberine-sensitive sensor were investigated. The sensor can be applied to the quantification of berberine with a linear range covering from 4.0×10⁻⁷ to 2.0×10⁻⁵ mol l⁻¹ with a detection limit of 8.0×10⁻⁸ mol l⁻¹. The sensor exhibits excellent reproducibility, reversibility and selectivity. The recommended method was successfully used for the determination of berberine in pharmaceutical preparations.     

The micro water/1,2-dichloroethane interface as a transducer for creatinine assay

Facilitated ammonium ion transfer reactions, using the crown ether dibenzo-18-crown-6, at a single microhole supported water/1,2-dichloroethane interface were used as the transducer to detect the biomolecule creatinine. The ammonium ion was produced during the enzymatic action of creatinine deiminase on creatinine. A prototype sensing device incorporating a Teflon gas permeable membrane, to exclude possible interferents, yielded a linear amperometric response to added creatinine from 20 M to 1 mM. This range is sufficient for that required for the determination of creatinine in serum.     

The use of an immobilized enzyme nylon tube reactor incorporating a four enzyme system for creatinine analysis

A single immobilized enzyme nylon tube reactor was produced incorporating a four enzyme system for the analysis of creatinine. The enzyme activity ratios in the coupling solution used to prepare the reactor were found to be of extreme importance in governing the activity of the latter. The reactor was incorporated into a continuous flow analysis system used to assay creatinine in urine samples and the results were correlated with a manual technique employing the same enzyme system in solution. The precision, correlation, high specificity, simplicity, and speed of the analysis were concluded to be factors in favor of the method's suitability for urine creatinine determinations.     

Biosensor for L-phenylalanine based on the optical detection of NADH using a UV light emitting diode

A biosensor was developed for the detection of L-phenylalanine (Phe) and demonstrated for use in the diagnosis of phenylketonuria (PKU). It consists of L-phenylalanine dehydrogenase (L-PheDH) immobilized on a membrane, an ultraviolet light-emitting diode excitation system, and a photomultiplier tube. The L-PheDH was immobilized on a teflon membrane modified with 2-methacryloyloxyethyl phosphorylcholine and placed at the distal and of an optical fiber. The concentration of Phe was determined by immersing the sensor into a sample solution that also contained NAD⁺ and measurement of the fluorescence of the NADH produced by enzymatic reaction. Two L-PheDHs (from Thermoactinomyces intermedius and Sporosarcina sp.) were studied and compared. The fluorescence intensities of the biosensor are linearly related to the L-Phe concentrations in the range from 10 μmol L^?1 to 10 mmol L^?1. The sensor also was operated in the kinetic mode by differential determination of the slope of the signal within 2 min. The analytical range of the sensor is adequate for application in the genotypic diagnosis of PKU (diagnostic value >600 μmol L^?1). High sensitivity, good cost-benefit ratio, and low power consumption are typical features of this biosensing system that can can be applied to routine screening of newborn.

Development of a Simple and Inexpensive Optical Absorption One‐Shot Sensor Membrane for Detection and Determination of Cyanide Ions in Water Samples

A new one‐shot optical cyanide ion sensor is proposed for determination of cyanide ions. The sensor was constructed by immobilizing crystal violet (CV) on triacetylcellulose membrane. The sensing mechanism involves reaction between cyanide ions and the immobilized CV at pH = 5.4, which results in a decrease in absorbance of the membrane at 600 nm. The sensor shows sufficient repeatability, reproducibility, operational lifetime of 3 weeks, and a response of less then 10 min under the optimum conditions and response time of 8 min. Cyanide can be determined in the concentration range of 50.0‐800 μg mL^‐1 with a detection limit of 5.0 μg mL^‐1. Most ions do not interfere with the determination of cyanide ions. The proposed sensor was successfully applied to the determination of cyanide in spiked water samples.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Determination of glutamate-pyruvate transaminase activity in blood serum with a pyruvate oxidase/poly(vinyl chloride) membrane sensor

Pyruvate oxidase (E.C. 1.2.3.3.) is immobilized by adsorption on a wet PVC membrane. Glutamate-pyruvate transaminase activity (5–1600 IU l^?1) in serum is determined by a pyruvate oxidase sensor consisting of the immobilized pyruvate oxidase coupled to a platinum electrode for measuring hydrogen peroxide, after an l-alanine—α-ketoglutarate reaction. The assay requires ?60 s, and has a precision of 2–3%. Endogenous pyruvate should not interfere if measurements are made > 30 s after starting the reaction.     

Grape tissue-based electrochemical sensor for the determination of hydrogen peroxide

The use of grape tissue as a source of catalase for the determination of hydrogen peroxide is reported. A slice of grape tissue attached to the membrane of a Clark-type oxgen sensor was used to monitor the oxidation of hydrogen peroxide by catalase. At the steady state, the sensor responds linearly to hydrogen peroxide in the concentration range 1 × 10^?5–5 × 10^?4 M. The response time (T₉₀) was of the order of 1 min for this sensor. No interference was observed from ethanol, amino acids, glucose and lactic acid. The long-term stability of the grape tissue sensor was much better than previously reported immobilized enzyme and liver tissue-based hydrogen peroxide sensors.