Enzyme-based sensors

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Another approach, developed in our laboratory, consists of the compartmentalization of the sensing layer25"27. This concept, only applicable for multi-enzyme based sensors, consist in immobilizing the luminescence enzymes and the auxiliary enzymes on different membranes and then in stacking these membranes at the sensing tip of the optical fibre sensor. This configuration results in an enhancement of the sensor response, compared with the case where all the enzymes are co-immobilized on the same membrane. This was due to an hyperconcentration of the common intermediate, i.e. the final product of the auxiliary enzymatic system, which is also the substrate of the luminescence reaction, in the microcompartment existing between the two stacked membranes. 

The one-substrate mechanism is frequently operative in enzyme-based sensors. However, when several substrates or two or more (coupled enzymatic reactions) are required for sensor performance, the derived equations are more complex and usually require simplification for solving35. 

Fuh M.R.S., Burgess L.W., Christian G.D., Single fiber-optic fluorescence enzyme-based sensor, Anal. Chem. 1988 60 433-435. 

In terms of enzyme-based sensors for fermentation monitoring, the main problem is the lack of enzyme stability during long-term use. Two recent reviews describe the current state of the art, 62,63) so we will not dwell on their use. 

Glucose sensors based on this electrochemistry are now commercially available. Furthermore, it seems likely that this concept will soon be expanded to other types of enzyme-based sensors. Hence, sensor development is proving to be one of the great success stories of the chemically modified electrode research area. 

Chapters 1 to 5 deal with ionophore-based potentiometric sensors or ion-selective electrodes (ISEs). Chapters 6 to 11 cover voltammetric sensors and biosensors and their various applications. The third section (Chapter 12) is dedicated to gas analysis. Chapters 13 to 17 deal with enzyme based sensors. Chapters 18 to 22 are dedicated to immuno-sensors and genosensors. Chapters 23 to 29 cover thick and thin film based sensors and the final section (Chapters 30 to 38) is focused on novel trends in electrochemical sensor technologies based on electronic tongues, micro and nanotechnologies, nanomaterials, etc. 

This book on Electrochemical (Bio)Sensor Analysis, edited by S. Alegret and A. Merko< i, is an additional step to advance the field of rapid analysis. It presents advanced sensor developments as well as practical applications of electrochemical (bio)sensors in various fields in a single source. The book contains 38 chapters grouped into seven sections (a) Potentiometric sensors, (b) Yoltammetric (bio)sensors, (c) Gas sensors, (d) Enzyme based sensors, (e) Affinity biosensors, (f) Thick and thin film biosensors, and (g) Novel trends. This interdisciplinary book has contributions from well-known specialists in the field and will be a useful resource for professionals with an interest in the application of electrochemical (bio)sensors. 

Part I contains general reviews on the theoretical and practical aspects related to the application of (bio) sensors in various fields. Its 38 chapters are grouped in seven sections 1) Potentiometric sensors, 2) Voltammetric sensors, 3) Gas sensors, 4) Enzyme based sensors, 5) Affinity biosensors, 6) Thick and thin film (bio) sensors and 7) Novel trends. 

Moreno-Bondi MC, Benito-Pena E (2005) Fundamentals of enzyme-based sensors. In Martellucci S, Baldini F (eds) Optical chemical sensors. Springer-Kluwer, New York (in press)... 

Curey TE, Goodey A, Tsao A, Lavigne J, Sohn Y, McDevitt JT, Anslyn EV, Neikirk D, Shear JB. Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array. Analytical Biochemistry 2001, 293, 178-184. 

SWNT-based glucose sensors an enzyme-based sensor and an affinity sensor. These results are a promising beginning however, there is much work left to do before the promise of SWNT in vivo sensors is realized. Investigation of new sensing modalities and optimization of current ones is necessary before the best nanotube-based sensing strategy is discovered. 

Alcohol oxidase. We have chosen to use alcohol oxidase from Hansenula polv-morpha (2) in our research. The enzyme has eight sub-units, all of which must be associated for the enzyme to exhibit activity. It is a large enzyme Mn 600,000 and has poor stability upon freeze drying. This factor combined with an almost flat activity response to pH between 6 and 10 and a maximum temperature activity at 40-50°C made the enzyme an ideal candidate for the study of enzyme stabilization in the dry state, especially as we wished to prepare diagnostic kits for ethanol and solid phase enzyme based sensors. 

Hu, Y., Wilson, G. S. A temporary local energy pool coupled to neuronal activity fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzyme-based sensor. /. Neurochem. 1997, 69 1484-1490. 

Enzyme-based sensors without catalytic transformation of the analyte... 

Enzyme-based sensors can be based on detecting hydrogen peroxide, oxygen, or H, depending on the analyte and enzyme. Voltammetric sensors are used for H2O2 and O2, while a potentio-metric pH electrode is used for H. ... 

Electrochemical biosensors have been divided into two basic types enzyme-based sensor and electrochemical probe-based sensor. Alkaline phosphatase (ALP) and horse radish peroxidase (HRP) have been often employed for enzyme-based biosensors using p-nitrophenyl phosphate (PNP), a-naphtyl phosphate, 3-3, 5,5 -tetramethylbenzidine (TMB) and 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrates of electrochemically active species, and ferrocene (Fc) and methylene blue as the electrochemical mediators. In general, enzymatic amplification of electrochemical signals enables highly sensitive detection of analytes. On the other hand, a direct detection of analytes by using electrochemical probes allows a more rapid time-response onto the detector surface and needs no enzymatic reaction. Based on the reason, a direct detection of analytes by using electrochemical probes has been... 

Probably the most important developments in amperometric sensors were the Clark oxygen electrode and the amperometric glucose sensor. The latter is the most successful example of an enzyme-based sensor. 

The kinetics of enzyme-catalyzed reactions can be very complex, and the mathematical representations for the effect of the concentrations of substrate, product, cofactors, and inhibitors are presented in a variety of textbooks in this field [1]. The exact form of this dependence of enzyme activity on these factors might have a profound effect on the behavior of an enzyme biosensor. However, one can delineate general rules of thumb concerning the properties of enzymes for the preliminary design of enzyme-based sensors. 

Response time of bioreceptor-based sensors. As in enzyme-based sensors the two main factors that determine the responsiveness of bioreceptor-based sensors are diffusive and kinetic phenomena. 

Suitable matrices for immobilization of biological reagents, especially enzymes, is an active area of biosensor research [7]. Because the activity of an enzyme is determined by its three-dimensional structure, and that structure is, in turn, related to the environment into which the enzyme is placed, the sensitivity of an enzyme-based sensor is greatly affected by this choice of immobilization matrix. 

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