Nylon enzyme electrodes

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

Among potentiometric enzyme sensors, the urea enzyme electrode is the oldest (and the most important). The original version consisted of an enzyme layer immobilized in a polyacrylamide hydrophilic gel and fixed in a nylon netting attached to a Beckman 39137 glass electrode, sensitive to the alkali metal and NHj ions [19, 2A Because of the poor selectivity of this glass electrode, later versions contained a nonactin electrode [20,22] (cf. p. 187) and especially an ammonia gas probe [25] (cf. p. 72). This type of urea electrode is suitable for the determination of urea in blood and serum, at concentrations from 5 to 0.05 mM. Figure 8.2 shows the dependence of the electrode response... 

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. 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

The above authors coimmobilized choline oxidase and AChE on a nylon net which was fixed to a hydrogen peroxide probe so that the esterase was adjacent to the solution. The apparent activities were 200-400 mU/cm2 for choline oxidase and 50-100 mU/cm2 for AChE. The sensitivity of the sequence electrode for ACh was about 90% of that for choline, resulting in a detection limit of 1 pmol/l ACh. The response time was 1-2 min. The parameters of this amperometric sensor surpass those of potentiometric enzyme electrodes for ACh (see Section 3.1.25). Application to brain extract analysis has been announced. 

A second approach to modification of electrode surfaces prior to their use in flowing streams has been to immobilize enzymes onto the surface. These techniques have long been used in the development of amperometric enzyme electrodes, but have only recently been applied to on-line analysis using (primarily) FIA. Some examples from the recent literature include the use of glucose oxidase immobilised onto either a Clark oxygen electrode or nylon net for the determination of glucose down to sub mM levels, and chymotrypsin... 

In our method [176] of enzyme electrode preparation the enzymes are immobilized on a partially hydrolyzed nylon net via Ugi s four-component reaction [177]. This reaction has been used in two different ways. In the first method the nylon net was partially hydrolyzed with hydrochloric acid and the enzymes were covalently bound on this activated net by the reaction with glutaraldehyde and cyclohexyl isocyanide. The existence of four amide bonds is the result of this reaction. The immobilization of enzymes by means of these amide bonds is more effective than via Shiff s bases which are produced in the most common method for enzyme electrode preparation. When glutaraldehyde alone was used for the immobilization, i.e., without cyclohexyl isocyanide, the resulting enzyme electrode showed an approximately fivefold lower activity. 

It is also possible to trap an enzyme to the sensitive component of an electrode using a dialysis membrane that prevents protein diffusion. Guibault and Shu [23] constructed a urea-sensitive enzyme electrode by spreading a suspension of urease over the surface of an electrode equipped with a nylon net, and covering the whole assembly with a dialysis membrane the enzyme electrode is then rinsed and is ready for use. 

The pOz transducer is useful because it provides a hydrophobic support onto which an enzyme can easily be fixed. Glucose oxidase can be immobilized trapped in a polyacrylamide gel [2, 142], coreticulated with albumin [3] or gelatine [143], or immobilized using a nylon net [113]. Enzyme electrodes obtained in this way have been used for the determination of glucose in blood and serum, and have also been tested for monitoring fermentation processes [ 144]. 

Mascini M., lannillo M. and Palleschi G. (1983) Enzyme electrodes with improved mechanical and analytical characteristics obtained by binding enzymes to nylon nets. Anal. Chim. Acta, 146, 135-148. 

In the previous papers(12,13), we reported on the vessel access type, i.e. tubular type, glucose sensor. It consisted of a glucose electrode system with a GOX enzyme immobilized Nylon membrane and a glucose semipermeable membrane, and a reference oxygen electrode system. The sensor could directly measure up to 700 mg/dl of BGL in an arterial blood stream when it was placed into an external A-V shunt. This sensor, however, has some problems such as thrombus during in vivo testing without heparin and clinical complexity associated with implanting the sensor in a blood stream. 

The scope of ion-selective electrodes (ISEs) has been greatly enhanced by employing a poly(vinyl chloride) matrix to entangle sensor cocktail materials. Fbr ISFET devices an in situ photopolymerisation of monobutyl methacrylate provides a viable poly(butyl methacrylate) calcium sensor film with good gate adhesion properties. One or more enzymes can be chemically immobilized on modified nylon mesh. The resultant matrices are suitable for the amperometric assay of carbohydrates in blood and food products. 

Thus, glucose oxidase can be randomly immobilised on the modified nylon mesh (I). The resultant enzyme membrane (II) when held tautly over a platinun anode disc provides a high performance, long life glucose electrode which can be housed in a Stelte cell adapted for flow injection analysis (4). 

The influence of different spacer and coupling molecules on the relative performances of seventeen glucose oxidase-nylon electrodes (NGO) fabricated from the same batch of fresh enzyme have been conveniently established in the FIA mode with standard glucose( 1 mM) ... 

Multienzyme tylon Electrodes. Di- and polysaccharides require more than one enzyme to realise the amperanetrically detectable hydrogen peroxide and even glucose really needs the back up of mutarotase with glucose oxidase. It is fortunate that all the necessary enzymes can be immobilized simultaneously on just one nylon net. Thus a viable starch electrode has been fabricated (6) from a nylon net immersed in a cocktail of glucose oxidase, mutarotase and amylogluoosidase (Figure 1). Its response to a continuous flow of 0.1% m/v starch remained steady for over a period of 60 h. 

Utilike the enzyme nylon meshes, the viscose acetate-catalase membrane became very brittle after a y-radiation dose of 1.2 Mr ad, a condition which often led to tearing when attempting to reassemble the electrode for recalibration (7). 

The first electrode for urea was prepared by immobilizing urease in a poly-acrylcimide gel on nylon or Dacron nets. The nets were placed onto a Beckman electrode (NH J selective) (59). In a later development, the electrode was improved by covering the enzyme gel layer with a cellophane membrane to prevent leaching of urease into the solution (60). The urease electrode could be used for 21 days with no loss of activity. 

Enzymes have been attached to a nylon matrix (135-13 7), a pig intestine (138), the hydrophobic membrane of a gas-selective sensor (139), and controlled pore glass (140). Recent comparative studies of the coupling agents GA and benzoquinone support the preferential use of benzoquinone for binding GOD to nylon mesh (13 7), or to cellulose acetate membranes (141) with lysine (13 7). Both investigations report robust electrode behaviors with respect to prolonged exposure to glucose, while lifetimes of the membrane electrodes were ca. 3 months. 

Cholesterol can be determined by immobilizing the enzyme cholesterol oxidase (COD) in a layer over an oxygen electrode or a hydrogen peroxide electrode. Cholesterol oxidase is chemically immobilized onto nylon net (135, 265) or collagen membranes (267) and fixed onto an O2 electrode ... 

An enzyme immunoelectrode suitable for the assay of human serum albumin and insulin uses an oxygen electrode covered with an antibody-containing nylon net kept in place with an O-ring. From 1 to 25 ng/L of albumin and 5 to 100 ng/L of insulin can be assayed (306). A specific sensor for the tumor antigen a-fetoprotein (AFP) is prepared by immobilizing anti-AFP antibody covalendy on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyl octane, and GA (307). The sensor is applied to an EIA based on competitive Ab/... 

The enzymes have been both physically entrapped in polyacrylamide on nylon netting and chemically bound to polyacrylic acid derivatives both preparations exhibited large measuring times. Improvement of the system in favour of the response time diminished the sensitivity of the sensor. The authors reported a response time between 77 and 235 s and a sensitivity of 40 mV per concentration decade. Besides the low selectivity of the iodide sensitive electrode (thiocyanate, sulfide, cyanide, and silver(I) ions interfere), disturbances by other HRP substrates, e.g. uric acid, ascorbic acid, and Fe(II) ions, restrict the applicability of the method. 

Cholesterol electrodes based on the registration of oxygen consumption have also been described. The enzyme was immobilized to collagen membranes by crosslinking with BSA and glutaraldehyde (Satoh et al., 1977) or bound to nylon (Mascini et al., 1983) and fixed in front of the O2 probe. The sensor developed by Satoh et al. has been applied to serum samples. 

The very same enzymes have been combined by Mascini et al. (1985a) in an FIA system. Creatinine iminohydrolase was immobilized on the inner wall of nylon tubing (diameter 1 mm, length 1 m) and the ammonia liberated in the enzymatic reaction was measured with an NH3 electrode. Owing to the low sensitivity of the indicator electrode, the linear range was only 0.01-0.2 mmolA. 

Encapsulation achieves the confinement of biological components by using various semi-permeable membranes. Encapsulation allows for the enzymes to exist freely in solution, which is confined within the small area surrounded by the membrane. Macromolecules cannot cross the membrane barrier, which is permeable for small molecules only (substrates or products). Nylon and cellulose nitrate are the most popular materials used for the production of microcapsules that need to have a chameter between 10 and 100 pm chameteis. Furthermore, biological cells could be used as capsules as it shown in erythrocytes based sensor. Alternatively enzyme solution can be encapsulated in a thin layer, which covers the electrode and confined between the electrode and semi-permeable membrane surface. ... 

In this case, the urease is physically entrapped in a polyacrylamide matrix polymerized on the surface of an ammonium-ion glass electrode. The enzyme-gel matrix is supported on the electrode by a sheer dacron or nylon gauze, about the thickness of a nylon stocking, or it is held by a thin semipermeable cellophane sheet. The urea diffuses to the urease-gel membrane, where it is hydroly d to produce ammonium ion. Some of the ammonium ion diffuses through the thin membrane to the electrode surface, where it is monitored by the ammonium-sensitive electrode. The urea electrode is fairly stable, sensitive, specific for urea, has a usable lifetime of 2-3 weeks before a new gel layer must be prepared, and has a fairly fast response time ( < 120 sec). The output of the electrode is linear from about 10 to 10" M urea. 

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