Enzyme immobilization matrix surface

Du et al. reported a sensitive, fast and stable amperometric sensor for quantitative determination of OP insecticide, triazophos [14], Where, AChE was immobilized on MWNTs-chitosan (MC) composite matrix. Prior to enzyme immobilization, GCE surface was activated by applying a potential of +1.75 V for 300 s and scanned in the potential range +0.3 to +1.25V and +0.3 to -1.3V until a steady-state curve was obtained. This pretreated GCE surface was coated with 2.0 pi of MWCNTs, chitosan and glutaraldehyde mixture, followed by coating 4 pi of AChE solution, dried and used. CV results show that the oxidation peak of thiocholine occurs at +0.66V with much higher peak height at AChE/MC/GCE than at AChE/CS/GCE without MWCNTs. This shows that MWCNTs presence lowers the oxidation potential of thiocholine at the MC composite electrode. CV studies were also carried out to study the inhibition activity of triazophos at the composite electrode. The results show that, the peak currents decreased at the composite electrode with increase in triazophos concentration (Fig. 4). 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

As already discussed, a covalent immobilization can be performed via different chemical moieties on the protein surface. Because of that, protein molecules are immobilized in random orientation with at least one, but often several, covalent bonds to the matrix. As a result, the active site might be oriented toward the matrix surface and its accessibility to the substrate molecule hence significantly reduced. This results in a decrease of biological activity and consequently in lower binding capacity or decrease of reaction rate in the case of enzymes. 

Membrane reactors can be considered passive or active according to whether the membrane plays the role of a simple physical barrier that retains the free enzyme molecules solubilized in the aqueous phase, or it acts as an immobilization matrix binding physically or chemically the enzyme molecules. Polymer- and ceramic-based micro- and ultrafiltration membranes are used, and particular attention has to be paid to the chemical compatibility between the solvent and the polymeric membranes. Careful, fine control of the transmembrane pressure during operation is also required in order to avoid phase breakthrough, a task that may sometimes prove difficult to perform, particularly when surface active materials are present or formed during biotransformahon. Sihcone-based dense-phase membranes have also been evaluated in whole-cell processes [55, 56], but... 

After immobilizing this enzyme on the surface of insoluble matrix by physical adsorption, it was found that the Ka% value was increased to 0.08 mol/L whereas the rfl ax value stayed the same as rmax. What is the effectiveness factor of the immobilized enzyme when the substrate concentration is 1 mol/L ... 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

Fig. 1 a and b. Summary of techniques for enzyme immobilization. An enzyme can be immobilized by fixing it on the surface of a macroscopic material or by trapping it inside a matrix which is permeable to the enzyme s substrate. (Reprinted from Bailey, J. E., Ollis, D. F. Biochemical Engineering Fundamentals, p. 184, New York-London-Tokyo, McGraw-Hill 1977)... 

For an immobilized enzyme it follows that a reduction in the rate of diffusion of a substrate to the active site of an enzyme will increase the apparent Km and reduce Fmax. The nature of the mass transfer effect depends on the fashion in which the enzyme is immobilized. Enzymes immobilized on the surface of a carrier will experience external mass transfer limitations between the bulk solution and the surface, whereas those entrapped within a porous matrix are also affected by internal mass transfer limitations due to the reduction in the rate of diffusion of substrate and products through the matrix. 

Extracorporeal dialysis can be implemented for blood decontamination. Enzymes can be immobilized on dialysis cartridges (Klein and Langer, 1986). In that case, kcJKm has to be as high as possible and the flow rate reduced to increase the efficiency of the reactor. Moreover, accessibility of OP molecules to the enzyme active center must not be altered by the immobilization method or by matrix effects. The enzyme concentration per surface unit has to be maximized to reduce diffusion constraints. First order... 

In this section, we discuss about the screen printed electrode (SPE) based AChE sensors for the selective determination of OP and CA pesticides. In the past decades, several attempts were made by the researchers to develop SPE based pesticide sensors, where the enzyme AChE was immobilized either directly onto the electrode or above other matrices incorporated SPE surfaces. Both approaches resulted in the good, rapid detection of OP and CA pesticides. Earlier, Hart et al. employed AChE/SPE to detect OP and CA pesticides [21], They measured the enzyme activity from the rate of hydrolysis of acetylthiocholine iodide. Three polymers such as hydroxyethyl cellulose, dimethylaminoethyl methacrylate, and polyethyleneimine were used as enzyme immobilization matrices. Initially, electrodes were exposed to drops of water or pesticide solution, dried and their activity was screened after 24 h. They found that, when the enzyme matrix was hydroxyethyl cellulose, electrode activity inhibited both by water as well as by pesticides. While with co-polymer matrix, a significant response towards pesticides alone was observed. Further, the long-term storage stability of electrodes was highest when the enzyme matrix consisted of the co-polymer. The electrodes retained their activity for nearly one year. In contrast, the electrodes made of hydroxyethyl cellulose or polyethyleneimine possess less stability. 

Other than AUNPs, Quantum dots have also been employed in the development of pesticide sensors. Li et al. have synthesized Poly (N-vinyl-2-pyrrolione) (PVP)-capped CdS quantum dots (QCdS-PVP) from CdC and Na2S in the presence of PVP [37], AChE was immobilized onto this QCdS-PVP matrix incorporated GCE surface. The resulting GCE/ QCdS-PVP/AChE sensor was used for the detection of OP pesticides, such as trichlorfon. The enzyme immobilization procedure was described as follows. About 3 ml of QCdS-PVP was deposited on the surface of the GCE and dried in air. Then 3 ml of 0.5 mg ml"1 AChE along with 2.5% GA was deposited on the surface of the QCdS-PVP modified GCE and dried for 1 h at room temperature. TEM results show that, the QCdS-PVP particles were homogeneously distributed and they possess an average size of 2-4 nm (Fig. 9). 

The reuse of an enzyme can be economically favorable when a high-cost enzyme is used. It can be difficult to separate and reuse an enzyme because enzymes are typically globular proteins that are highly soluble in water. A common technique to facilitate the reuse of a high-value enzyme is to immobilize the enzyme onto a surface, inside of an insoluble matrix or within a semipermeable membrane. Both chemical and physical means can be employed to immobilize enzymes. The former method involves the covalent attachment of enzymes to water-insoluble supports and is the most widely used method for enzyme immobilization. ... 

Lee outlines three different physical methods that are commonly utilized for enzyme immobilization. Enzymes can be adsorbed physically onto a surface-active adsorbent, and adsorption is the simplest and easiest method. They can also be entrapped within a cross-linked polymer matrix. Even though the enzyme is not chemically modified during such entrapment, the enzyme can become deactivated during gel formation and enzyme leakage can be problematic. The microencapsulation technique immobilizes the enzyme within semipermeable membrane microcapsules by interfacial polymerization. All of these methods for immobilization facilitate the reuse of high-value enzymes, but they can also introduce external and internal mass-transfer resistances that must be accounted for in design and economic considerations. 

Figure 3.1 is a scanning electron microscopy (SEM) photograph of Novozym 435 before and after immobilization of CALB on the matrix (Lewatit). It is obvious that after immobilization, the enzyme has been adsorbed on the surface of the matrix and the surface has been saturated. This observation confirms the results of synchrotron infrared microspectroscopy performed at amide band wavelength on Novozym 435 (Figure 3.2) [5, 6], The researchers measured the intensity of the amide band across the cross-section of a Novozym 435 bead and attributed the presence of amide groups to the location of the enzyme immobilized on the bead. They showed that distribution of CALB on the bead is not homogenous and it mostly saturates the surface of the beads and hardly enters the center. The CALB enzyme is a globular protein with dimensions of 30 A x 40 A x 50 A [10], whereas...

The nested packed reactor (Fig. 5A0d) allows sample pretreatment prior to injection by means of solid oxidants, reductants, ion exchangers, immobilized enzymes, or suitable surface-active sorbents. The potential of this approach is largely unexploited, since so far such sample pretreatment has been used only to remove unwanted matrix, which is not retained on the column, for sample preconcentration, and for analyte conversion in connection with AAS and ICP (cf. Chapter 4.7). 

Enzyme-Substrate Electrodes. Electrodes that can respond to a variety of organic and biological compounds are constructed by coating the surface of an appropriate ion-selective electrode with an enzyme immobilized in some matrix. Perhaps the most well-known of these is the urea electrode [10], which makes use of the enzyme urease to hydrolyze urea (the substrate ) ... 

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