Phosphate enzyme sensor

An example of an optical enzyme sensor (Arnold, 1985) in a bifurcated optical fiber is shown in Fig. 9.32. The bifurcated fiber delivers and collects light to and from the site of the enzymatic reaction. The enzyme, alkaline phosphatase (AP), catalyzes hydrolysis of p-nitrophenyl phosphate to p-nitrophenoxide ion which is being detected (A = 404 nm). 

Characteristics of selected enzyme sensors for phosphate determination... 

E. Watanabe, S. Tokimatsu, and K. Toyama, Simultaneous Determination of Hypoxanthine, Inosine, Inosine-5 -Phosphate and Adenosine-5 -Phosphate with a Multielectrode Enzyme Sensor. Anal. Chim. Acta, 164 (1984) 139. 

Conrath, N. et al., A novel enzyme sensor for the deterrmination of inorganic phosphate, Analytica Chimica Acta 309, 47,1995. 

Watanabe E., Endo H. and Toyama K. (1988) Determination of phosphate ions with an enzyme sensor system. Biosensors, 3, 297-306. 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

Anions play key roles in chemical and biological processes. Many anions act as nucleophiles, bases, redox agents or phase transfer catalysts. Most enzymes bind anions as either substrates or cofactors. The chloride ion is of special interest because it is crucial in several phases of human biology and in disease regulation. Moreover, it is of great interest to detect anionic pollutants such as nitrates and phosphates in ground water. Design of selective anion molecular sensors with optical or electrochemical detection is thus of major interest, however it has received much less attention than molecular sensors for cations. 

The carrier used for this purpose consisted of a 0.1 M phosphate buffer of pH 7. The appearance of the sensing microzone is shown in Fig. 5.5.B. The oxygen optrode used was based on a 10-pm silicone rubber film containing dissolved decacyclene as indicator (S) that was fixed on a 110-pm thick polyester support (PS). A 9-pm black PTFE membrane (I) was used for optical insulation. The dye fluorescence was found to be markedly dependent on the concentration of oxygen, which exerted a quenching effect on it. The enzyme (glutamate oxidase) was immobilized on a 150-pm thick immunoaffmity membrane (E). The sensor was prepared similarly as reported by Trettnak et al. [7]. 

S ATP -I- thiamine phosphate <1, 2> (<2> enzyme plays an important role in the thiamine diphosphate biosynthetic pathway, thiamine diphosphate is the regulatory molecule for thiamine synthesis and predicts the existence of a sensor/regulatory protein [2]) (Reversibility <1, 2> [1, 2])... 

Sensors of this type were constructed as multi-electrode arrays bearing chohnesterases of differing sensitivity to organo-phosphates, and the assay extended to milk [35]. Both spiked milk and milk from shops inhibited the activity of the electrodes. In two instances, estimates of the level of organo-phosphates in spiked milk made using sensors were very close to those made using GC-MS. This appears to have been a fortunate co-incidence as the response curves used for calibration were not linear. Nine out of ten milk samples from shops inhibited at least some members of the array. In only one case did the GC-MS assay find insecticides in the samples, but the insecticides were not organo-phosphates. The inhibition shown by the enzyme-based arrays was reversible by pyridine-2-aldoxime... 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Phosphate is another anion which is of biological significance as well as being biologically active. Numerous phosphate biosensors have been developed, with one based on a multi-enzyme system immobilised on a cellulose membrane on a Pt electrode being able to detect levels down to 10 8 M [104]. Simpler methods, however, use enzymes such as polyphenol oxidase combined with alkaline phosphatase bound within an electropolymerised layer based on a substituted pyrrole [ 105]. This was reported to give a sensor with a detection... 

Sterilization of tylon Enzyme Electrodes. Sterilization of in vivo electrodes is essential for clinical use and advisable for applica-tions in the food industry. It was thus of interest to study the behaviour of various carbohydrate sensor membranes before and after irradiation with 60Co-y radiation (Figure 1). Thus, after each membrane calibration in a batch mode, the membrane was detached, placed in a sealed glass tube with phosphate buffer (pH 7) and irradiated to set doses. The membrane was then reattached to the... 

Hale et al. reported the use of an enzyme-modified carbon paste for the determination of acetylcholine [21], The sensor was constructed from a carbon paste electrode containing acetylcholineesterase and choline oxidase, and the electron transfer mediator tetrathiafulvalene. The electrode was used for the cyclic voltammetric determination of acetylcholine in 0.1 M phosphate buffer at +200 mV versus saturated calomel electrode. Tetrathiafulvalene efficiently re-oxidized the reduced flavin adenine dinucleotide centers of choline oxidase. The calibration graph was linear up to 400 pM acetylcholine, and the detection limit was 0.5 pM. 

Acetylcholineesterase Enzyme was covalently immobilized on a bovine serum albumin-modified H +-selective coated wire electrode. The sensor was used in 5mM phosphate buffer pH 7 at 30°C. The response time was 3-10 min for O.l-lOmM ACh. pH change of 6-8 had little effect. Coefficient of variation was 5.7 and 5.8%. [72]... 

Acetylcholineesterase and choline oxidase 300 pL 0.1 M phosphate buffer (pH 6.5) containing 16 mg BSA and 1 mg each of ChO and AChE were mixed with 30 pL 25% glutaral-dehyde diluted 10 fold with phosphate buffer. The solution was used to coat the surface of a Pt electrode. This enzyme electrode was used for the amperometric measurement of ACh and Ch. Calibration graphs were linear upto 0.09 and 0.08 mM Ch and ACh, respectively. Detection limits were 0.1 pM of both Ch and ACh. Response time was 1 s for both Ch and ACh. The use of the sensor as detector for HPLC analysis for both Ch and ACh was demonstrated. [91]... 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

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